U.S. patent application number 14/522360 was filed with the patent office on 2015-06-11 for compositions comprising an anti-pdgf aptamer and a vegf antagonist.
The applicant listed for this patent is OPHTHOTECH CORPORATION. Invention is credited to BYEONG SEON CHANG, RICHARD EVERETT.
Application Number | 20150157709 14/522360 |
Document ID | / |
Family ID | 49670532 |
Filed Date | 2015-06-11 |
United States Patent
Application |
20150157709 |
Kind Code |
A1 |
EVERETT; RICHARD ; et
al. |
June 11, 2015 |
Compositions Comprising an Anti-PDGF Aptamer and a VEGF
Antagonist
Abstract
The present invention is directed to compositions comprising an
anti-PDGF aptamer and a VEGF antagonist. In certain embodiments,
the compositions of the invention are useful for treating or
preventing an ophthalmological disease.
Inventors: |
EVERETT; RICHARD;
(PRINCETON, NJ) ; CHANG; BYEONG SEON; (SHERMAN
OAKS, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OPHTHOTECH CORPORATION |
NEW YORK |
NY |
US |
|
|
Family ID: |
49670532 |
Appl. No.: |
14/522360 |
Filed: |
October 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13797821 |
Mar 12, 2013 |
|
|
|
14522360 |
|
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|
|
61654672 |
Jun 1, 2012 |
|
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Current U.S.
Class: |
424/134.1 ;
424/133.1 |
Current CPC
Class: |
A61K 31/711 20130101;
A61K 31/711 20130101; A61K 47/26 20130101; C07K 2317/76 20130101;
C07K 2319/30 20130101; A61K 2300/00 20130101; A61K 9/0048 20130101;
A61K 2039/505 20130101; A61K 39/3955 20130101; A61K 45/06
20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/711 20060101 A61K031/711 |
Claims
1.-49. (canceled)
50. A method for preparing a composition, comprising: (a) admixing:
(i) Antagonist A; (ii) a VEGF antagonist selected from ranibizumab,
bevacizumab and aflibercept; and (iii) an effective amount of a
buffer; and optionally an effective amount of one or more of: (iv)
a tonicity agent; (v) a surfactant; (vi) a stabilizer; (vii) a
cryoprotectant; and (vii) a lyoprotectant; and (b) adjusting the pH
of the resulting mixture to a pH of about 5.5 to about 8.0.
51. The method of claim 1, wherein the VEGF antagonist is
ranibizumab.
52. The method of claim 1, wherein the VEGF antagonist is
bevacizumab.
53. The method of claim 1, wherein the VEGF antagonist is
aflibercept.
54. The method of claim 1, wherein the buffer is a Tris buffer, a
phosphate buffer, a histidine buffer or an acetate buffer.
55. The method of claim 1, wherein the tonicity agent is sodium
chloride or sorbitol.
56. The method of claim 1, wherein the surfactant is a
polysorbate.
57. The method of claim 1, wherein the stabilizer is a sugar, an
amino acid, a polyol, a surfactant, an antioxidant, a preservative,
a cyclodextrin, a polyethyleneglycol, albumin or a salt.
58. The method of claim 1, wherein the cryoprotectant is sucrose,
trehalose or glycerol.
59. The method of claim 1, wherein the lyoprotectant is sucrose,
trehalose or mannitol.
60. The method of claim 1, wherein the concentration of the nucleic
acid portion of Antagonist A is about 3 mg/mL; the VEGF antagonist
is bevacizumab, and the concentration of bevacizumab is about 12.5
mg/mL; the buffer is a phosphate buffer, and the concentration of
the phosphate buffer is about 50 mM; the composition comprises a
tonicity agent that is sodium chloride, and the concentration of
the sodium chloride is about 130 mM; the composition comprises a
surfactant that is polysorbate 20, and the concentration of the
polysorbate 20 is about 0.02% (w/v); and the pH of the composition
is about 6.0.
61. The method of claim 1, wherein the concentration of the nucleic
acid portion of Antagonist A is about 3 mg/mL; the VEGF antagonist
is ranibizumab, and the concentration of ranibizumab is about 5
mg/mL; the buffer is a histidine buffer, and the concentration of
the histidine buffer is about 10 mM; the composition comprises a
tonicity agent that is sodium chloride, and the concentration of
the sodium chloride is about 130 mM; the composition comprises a
surfactant that is polysorbate 20, and the concentration of the
polysorbate 20 is about 0.02% (w/v); and the pH of the composition
is about 6.0.
62. The method of claim 1, wherein the concentration of the nucleic
acid portion of Antagonist A is about 6 mg/mL; the VEGF antagonist
is aflibercept, and the concentration of aflibercept is about 40
mg/mL; the buffer is a phosphate buffer, and the concentration of
the phosphate buffer is about 10 mM; the composition comprises a
tonicity agent that is sodium chloride, and the concentration of
the sodium chloride is about 40 mM; the composition comprises a
cryoprotectant or lycoprotectant that is sucrose, and the
concentration of the sucrose is about 5% (w/v); the composition
comprises a surfactant that is polysorbate 20, and the
concentration of the polysorbate 20 is about 0.03% (w/v); and the
pH of the composition is about pH 6.2.
63. The method of claim 1, wherein at least about 80% of the
Antagonist A or VEGF antagonist of the composition shows no sign of
decomposition or modification resulting in formation of a new
chemical entity when stored at about room temperature for at least
two weeks.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 13/797,821, filed Mar. 12, 2013, which claims the benefit of
U.S. provisional application No. 61/654,672, filed Jun. 1, 2012,
the disclosure of each of which is incorporated by reference herein
in its entirety.
SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
OPHT.sub.--010.sub.--03US_ST25.txt. The text file is about 35 KB,
was created on Oct. 22, 2014, and is being submitted electronically
via EFS-Web.
FIELD OF THE INVENTION
[0003] This invention relates to compositions comprising an
anti-platelet derived growth factor (anti-PDGF) aptamer and a
vascular endothelial growth factor (VEGF) antagonist. This
invention also relates to methods for inhibiting hyperproliferation
of cells or aberrant angiogenesis, as well as to methods for
treating or preventing an ophthalmological disease, comprising
administering a composition comprising an anti-PDGF aptamer and a
VEGF antagonist. Furthermore, this invention relates to
compositions and drug delivery devices that provide extended
delivery of anti-PDGF aptamers and VEGF antagonists.
BACKGROUND OF THE INVENTION
[0004] Various disorders of the eye are characterized by, caused
by, or result in choroidal, retinal or iris neovascularization, or
retinal edema. These disorders include macular degeneration,
diabetic retinopathy, hypertensive retinopathy, central serous
chorioretinopathy, cystoid macular edema. Coats disease, and ocular
or adnexal neoplasms, such as choroidal hemangioma, retinal pigment
epithelial carcinoma, and intraocular lymphoma. Age-related macular
degeneration (AMD) is a disease that affects approximately one in
ten Americans over the age of 65. One type of AMD, "wet AMD," also
known as "neovascular AMD" and "exudative AMD." accounts for only
10% of AMD cases but results in 90% of cases of legal blindness
from macular degeneration in the elderly. Diabetic retinopathy can
affect up to 80% of all patients having diabetes for 10 years or
more and is the third leading cause of adult blindness, accounting
for almost 7% of blindness in the USA.
[0005] Advances have been made in understanding the molecular
events accompanying or leading to ocular neovascularization,
including the role of growth factors such as platelet derived
growth factor (PDGF) and vascular endothelial growth factor (VEGF).
Therapeutic agents that inhibit the activity of these growth
factors have been shown to provide a therapeutic benefit to
patients suffering from vascular disorders of the eye such as AMD
and diabetic retinopathy, including aptamers composed of synthetic
oligonucleotides. More recently, the combined use of therapeutic
agents that target either PDGF or VEGF is being explored.
[0006] Combined inhibition of both PDGF and VEGF may lead to a
greater benefit in treating various disorders of the eye that are
characterized by, caused by, or result in choroidal, retinal or
iris neovascularization, or retinal edema. Combined inhibition of
both PDGF and VEGF by individual agents specific to each growth
factor may be accomplished by simultaneous coadministration of both
agents.
[0007] Unfortunately, polypeptide therapeutic agents can be
susceptible to physical and chemical degradation. The stability of
polypeptide therapeutic agents can be influenced by a variety of
factors, including the polypeptide itself, e.g., its amino acid
sequence. Thus, the development of stable pharmaceutical
compositions comprising polypeptide therapeutics poses a
significant challenge. The challenge is even greater for the
development of compositions comprising a polypeptide therapeutic
and another therapeutic agent, such as a polynucleotide therapeutic
agent, since it requires the identification of excipients and
conditions that stabilize two different therapeutic agents with
acceptable compatability.
[0008] There is clearly a need in the art for stable compositions
comprising multiple therapeutic agents, including those comprising
an anti-PDGF aptamer and a VEGF antagonist.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides compositions comprising an
effective amount of: (a) an anti-PDGF aptamer or a pharmaceutically
acceptable salt thereof; and (b) a VEGF antagonist or a
pharmaceutically acceptable salt thereof. A composition comprising
an effective amount of (a) an anti-PDGF aptamer or a
pharmaceutically acceptable salt thereof and (b) a VEGF antagonist
or a pharmaceutically acceptable salt thereof is a "composition of
the invention."
[0010] In certain embodiments, a composition of the invention
comprises an effective amount of: (a) about 0.3 mg/mL to about 30
mg/mL Antagonist A or a pharmaceutically acceptable salt thereof;
(b) about 0.5 mg/mL to about 20 mg/mL ranibizumab or a
pharmaceutically acceptable salt thereof; and one or both of: (c) a
buffer capable of achieving or maintaining the pH of the
composition at about pH 5.0 to about pH 8.0; and (d) a tonicity
modifier. In certain embodiments, the buffer is about 1 mM to about
20 mM L-histidine or about 1 mM to about 20 mM sodium phosphate,
and the tonicity modifier is about 10 mM to about 200 mM NaCl,
about 1% to about 20% (w/v) sorbitol, or about 1% to about 20%
(w/v) trehalose. In particular embodiments, the composition of the
invention further comprises: (e) about 0.001% (w/v) to about 0.05%
(w/v) surfactant.
[0011] In certain embodiments, a composition of the invention
comprises an effective amount of: (a) about 0.3 mg/mL to about 30
mg/mL Antagonist A or a pharmaceutically acceptable salt thereof;
and (b) about 0.5 mg/mL to about 25 mg/mL bevacizumab or a
pharmaceutically acceptable salt thereof; and one or both of: (c) a
buffer capable of achieving or maintaining the pH of the
composition at about pH 5.0 to about pH 8.0; and (d) a tonicity
modifier. In certain embodiments, the buffer is about 5 mM to about
200 mM sodium phosphate or about 5 mM to about 200 mM Tris.HCl, and
the tonicity modifier is about 10 mM to about 200 mM NaCl, about 1%
to about 20% (w/v) sorbitol, or about 1% to about 20% (w/v)
trehalose. In particular embodiments, the composition of the
invention further comprises: (e) about 0.001% (w/v) to about 0.05%
(w/v) surfactant.
[0012] In certain embodiments, a composition of the invention
comprises an effective amount of: (a) about 0.3 mg/mL to about 30
mg/mL Antagonist A or a pharmaceutically acceptable salt thereof;
(b) about 5 mg/mL to about 40 mg/mL aflibercept or a
pharmaceutically acceptable salt thereof; and one or more of: (c) a
buffer capable of achieving or maintaining the pH of the
composition at about pH 5.0 to about pH 8.0, (d) a tonicity
modifier, and (e) 0 to about 10% (w/v) sucrose. In certain
embodiments, the buffer is about 5 mM to about 50 mM phosphate, and
the tonicity modifier is about 10 mM to about 200 mM NaCl. In
particular embodiments, the composition of the invention further
comprises: (f) about 0.001% (w/v) to about 0.05% (w/v)
surfactant.
[0013] In certain embodiments, a composition of the invention
comprises an effective amount of: (a) about 3 mg/mL to about 90
mg/mL Antagonist A or a pharmaceutically acceptable salt thereof;
(b) about 1.0 mg/mL to about 30 mg/mL ranibizumab or a
pharmaceutically acceptable salt thereof; and one or both of: (c) a
buffer capable of achieving or maintaining the pH of the
composition at about pH 5.0 to about pH 8.0; and (d) a tonicity
modifier. In certain embodiments, the buffer comprises about 1 mM
to about 100 mM sodium phosphate or about 1.0 mM to about 10 mM
histidine.HCl, and the tonicity modifier is about 0.5% (w/v) to
about 10% (w/v) trehalose.
[0014] The present invention further provides methods for treating
or preventing an ophthalmological disease, comprising administering
to a mammal in need thereof a composition of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows AEX-HPLC chromatograms of selected compositions
of the invention stored for 8 weeks at 37.degree. C.
[0016] FIG. 2 shows WCX-HPLC chromatograms of selected compositions
of the invention stored for 8 weeks at 37.degree. C.
[0017] FIG. 3 shows SE-HPLC chromatograms of selected compositions
of the invention stored for 8 weeks at 37.degree. C.
[0018] FIG. 4 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
37.degree. C.
[0019] FIG. 5 shows a WCX-HPLC trend graph of ranibizumab stability
in selected compositions of the invention stored at 37.degree.
C.
[0020] FIG. 6 shows a SE-HPLC trend graph of Antagonist A stability
in selected compositions of the invention stored at 37.degree.
C.
[0021] FIG. 7 shows a SE-HPLC trend graph of ranibizumab stability
in selected compositions of the invention stored at 37.degree.
C.
[0022] FIG. 8 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C.
[0023] FIG. 9 shows a WCX-HPLC trend graph of ranibizumab stability
in selected compositions of the invention stored at 25.degree.
C.
[0024] FIG. 10 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C.
[0025] FIG. 11 shows a SE-HPLC trend graph of ranibizumab stability
in selected compositions of the invention stored at 25.degree.
C.
[0026] FIG. 12 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
4.degree. C.
[0027] FIG. 13 shows a WCX-HPLC trend graph of ranibizumab
stability in selected compositions of the invention stored at
4.degree. C.
[0028] FIG. 14 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
4.degree. C.
[0029] FIG. 15 shows a SE-HPLC trend graph of ranibizumab stability
in selected compositions of the invention stored at 4.degree.
C.
[0030] FIGS. 16A and 16B show AEX-HPLC trend graphs of Antagonist A
stability in selected compositions of the invention having various
pHs stored at 37.degree. C. FIG. 16A shows the percent purity of
Antagonist A in compositions comprising 5% sorbitol over time at
various pHs, and FIG. 16B shows the percent purity of Antagonist A
in compositions comprising 130 mM NaCl over time at various
pHs.
[0031] FIGS. 17A and 17B show WCX-HPLC trend graphs of ranibizumab
stability in selected compositions having various pHs stored at
37.degree. C. FIG. 17A shows the percent purity of ranibizumab in
compositions comprising 5% sorbitol over time at various pHs, and
FIG. 17B shows the percent purity of ranibizumab in compositions
comprising 130 mM NaCl over time at various pHs.
[0032] FIG. 18 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions having various pHs stored at
37.degree. C.
[0033] FIGS. 19A and 19B show SE-HPLC trend graphs of ranibizumab
stability in selected compositions of the invention having various
pHs stored 37.degree. C. FIG. 19A shows the percent purity of
ranibizumab in compositions comprising 5% sorbitol, and FIG. 19B
shows the percent purity of ranibizumab in compositions comprising
130 mM NaCl.
[0034] FIG. 20 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention comprising
various tonicity modifiers at various pHs stored at 37.degree.
C.
[0035] FIG. 21 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention comprising
various tonicity modifiers at pH 8.0 stored at 37.degree. C.
[0036] FIG. 22 shows a WCX-HPLC trend graph of ranibizumab
stability in selected compositions of the invention comprising
various tonicity modifiers at various pHs stored at 37.degree.
C.
[0037] FIG. 23 shows a SE-HPLC trend graph of ranibizumab stability
in selected compositions of the invention comprising various
tonicity modifiers at various pHs stored at 37.degree. C.
[0038] FIG. 24 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
37.degree. C.
[0039] FIGS. 25A and 25B show AEX-HPLC trend graphs of Antagonist A
stability in selected composition of the invention stored at
25.degree. C. (FIG. 25A) and 37.degree. C. (FIG. 25B).
[0040] FIGS. 26A and 26B show WCX-HPLC trend graphs of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C. (FIG. 26A) and 37.degree. C. (FIG. 26B).
[0041] FIGS. 27A, 27B, and 27C show SE-HPLC chromatograms of
selected compositions of the invention stored for 8 weeks at
37.degree. C. (FIG. 27A), 25.degree. C. (FIG. 27B) and 4.degree. C.
(FIG. 27C).
[0042] FIG. 28 shows an AEX-HPLC trend graph of Antagonist A
stability in composition F6 stored at 4.degree. C., 25.degree. C.
and 37.degree. C.
[0043] FIG. 29 shows a WCX-HPLC trend graph of ranibizumab
stability in composition F6 stored at 4.degree. C., 25.degree. C.
and 37.degree. C.
[0044] FIG. 30 shows a SE-HPLC trend graph of Antagonist A
stability in composition F6 stored at 4.degree. C., 25.degree. C.
and 37.degree. C.
[0045] FIG. 31 shows a SE-HPLC trend graph of ranibizumab stability
in selected compositions of the invention stored at 4.degree. C.,
25.degree. C. and 37.degree. C.
[0046] FIG. 32 shows AEX-HPLC chromatograms of selected
compositions of the invention stored for two weeks at 37.degree.
C.
[0047] FIG. 33 shows WCX-HPLC chromatograms of selected
compositions of the invention stored for 8 weeks at 25.degree.
C.
[0048] FIG. 34 shows SE-HPLC chromatograms of selected compositions
of the invention stored for 8 weeks at 37.degree. C.
[0049] FIG. 35 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
37.degree. C.
[0050] FIG. 36 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
37.degree. C.
[0051] FIG. 37 shows a WCX-HPLC trend graph of bevacizumab
stability in selected compositions of the invention stored at
37.degree. C.
[0052] FIG. 38 shows a WCX-HPLC trend graph of bevacizumab
stability in selected compositions of the invention stored at
37.degree. C.
[0053] FIG. 39 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
37.degree. C.
[0054] FIG. 40 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
37.degree. C.
[0055] FIG. 41 shows a SE-HPLC trend graph of bevacizumab stability
in selected compositions of the invention stored at 37.degree.
C.
[0056] FIG. 42 shows a SE-HPLC trend graph of bevacizumab stability
in selected compositions of the invention stored at 37.degree.
C.
[0057] FIG. 43 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C.
[0058] FIG. 44 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C.
[0059] FIG. 45 shows a WCX-HPLC trend graph of bevacizumab
stability in selected compositions of the invention stored at
25.degree. C.
[0060] FIG. 46 shows a WCX-HPLC trend graph of bevacizumab
stability in selected compositions of the invention stored at
25.degree. C.
[0061] FIG. 47 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C.
[0062] FIG. 48 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
25.degree. C.
[0063] FIG. 49 shows a SE-HPLC trend graph of bevacizumab stability
in selected compositions of the invention stored at 25.degree.
C.
[0064] FIG. 50 shows a SE-HPLC trend graph of bevacizumab stability
in selected compositions of the invention stored at 25.degree.
C.
[0065] FIG. 51 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
4.degree. C.
[0066] FIG. 52 shows a WCX-HPLC trend graph of bevacizumab
stability in selected compositions of the invention stored at
4.degree. C.
[0067] FIG. 53 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
4.degree. C.
[0068] FIG. 54 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention stored at
4.degree. C.
[0069] FIG. 55 shows a SE-HPLC trend graph of bevacizumab stability
in selected compositions of the invention stored at 4.degree.
C.
[0070] FIG. 56 shows an AEX-HPLC trend graph of Antagonist A
stability in selected sorbitol-containing compositions of the
invention having various pHs stored at 37.degree. C.
[0071] FIG. 57 shows a WCX-HPLC trend graph of bevacizumab
stability in selected sorbitol-containing compositions of the
invention having various pHs stored at 37.degree. C.
[0072] FIG. 58 shows a SE-HPLC trend graph of Antagonist A
stability in selected sorbitol-containing compositions of the
invention having various pHs stored at 37.degree. C.
[0073] FIG. 59 shows a SE-HPLC trend graph of bevacizumab stability
in selected sorbitol-containing compositions of the invention
having various pHs stored at 37.degree. C.
[0074] FIGS. 60A and 60B show AEX-HPLC trend graphs of Antagonist A
stability in selected compositions of the invention having various
pHs stored at 37.degree. C. FIG. 60A shows the percent purity of
Antagonist A in compositions comprising 5% sorbitol over time at
various pHs, and FIG. 60B shows the percent purity of Antagonist A
in compositions comprising 130 mM NaCl or 150 mM NaCl over time at
various pHs.
[0075] FIGS. 61A and 61B show WCX-HPLC trend graphs of bevacizumab
stability in selected compositions of the invention having various
pHs stored at 37.degree. C. FIG. 61A shows the percent purity of
bevacizumab in compositions comprising 5% sorbitol, and FIG. 61B
shows the percent purity of bevacizumab in compositions comprising
130 mM NaCl or 150 mM NaCl over time at various pHs.
[0076] FIGS. 62A and 62B show SE-HPLC trend graphs of Antagonist A
stability in selected compositions of the invention having various
pHs stored at 37.degree. C. FIG. 62A shows the percent purity of
Antagonist A in compositions comprising 5% sorbitol, and FIG. 62B
shows the percent purity of Antagonist A in compositions comprising
130 mM NaCl or 150 mM NaCl over time at various pHs.
[0077] FIGS. 63A and 63B show SE-HPLC trend graphs of bevacizumab
stability in selected compositions of the invention having various
pHs stored at 37.degree. C. FIG. 63A shows the percent purity of
Antagonist A in compositions comprising 5% sorbitol, and FIG. 63B
shows the percent purity of Antagonist A in compositions comprising
130 mM NaCl or 150 mM NaCl over time at various pHs.
[0078] FIG. 64 shows an AEX-HPLC trend graph of Antagonist A
stability in selected compositions of the invention comprising
various concentrations of Antagonist A stored for 8 weeks at
37.degree. C.
[0079] FIG. 65 shows a WCX-HPLC trend graph of bevacizumab
stability in selected compositions of the invention comprising
various concentrations of Antagonist A stored for 8 weeks at
37.degree. C.
[0080] FIG. 66 shows a SE-HPLC trend graph of Antagonist A
stability in selected compositions of the invention comprising
various concentrations of Antagonist A stored at 37.degree. C.
[0081] FIG. 67 shows a SE-HPLC trend graph of bevacizumab stability
in selected compositions of the invention comprising various
concentrations of Antagonist A stored for 8 weeks at 37.degree.
C.
[0082] FIG. 68 shows an AEX-HPLC trend graph of Antagonist A
stability in composition F19 at various storage temperatures.
[0083] FIG. 69 shows a WCX-HPLC trend graph of bevacizumab
stability in composition F19 at various storage temperatures.
[0084] FIG. 70 shows a SE-HPLC trend graph of Antagonist A
stability in composition F19 at various storage temperatures.
[0085] FIG. 71 shows a SE-HPLC trend graph of bevacizumab stability
in composition F19 at various storage temperatures.
[0086] FIG. 72 shows an AEX-HPLC trend graph of Antagonist A
stability in composition F19 as compared to composition F25 at
various storage temperatures.
[0087] FIG. 73 shows a SE-HPLC trend graph of Antagonist A
stability in composition F19 as compared to composition F25 at
various storage conditions.
[0088] FIG. 74 shows a WCX-HPLC trend graph of bevacizumab
stability in composition F19 as compared to composition F18 at
various storage temperatures.
[0089] FIG. 75 shows a SE-HPLC trend graph of bevacizumab stability
in composition F19 as compared to composition F18 at various
storage conditions.
[0090] FIG. 76 shows a graph depicting suppression of VEGF-induced
TF expression by various compositions of the invention.
[0091] FIG. 77 shows a graph depicting suppression of PDGF-induced
BTG2 expression by various compositions of the invention.
[0092] FIGS. 78A-F shows the structure of Antagonist A, where
designations {circle around (B)}-{circle around (F)} indicate a
continuation from a previous panel.
[0093] FIGS. 79A and 79B show graphs depicting the subtracted
micro-flow imaging (MFI) results for Composition F27 under varying
storage conditions. The graphs provide the particle count (number
of particles/mL) determined for each of the listed equivalent
circular diameter ranges when stored at either 5.degree. C. or
30.degree. C. in either a vial or a syringe. FIG. 79A provides the
particle counts within various ranges spanning 1 .mu.m to 100 .mu.m
equivalent circular diameter, and FIG. 79B provides the particle
counts within selected ranges spanning 10 .mu.m to 100 .mu.m
equivalent circular diameter. The legends from top to bottom
correspond to the bars from left to right for each particle
diameter range.
[0094] FIGS. 80A and 80B show graphs depicting the subtracted MFI
results for Composition F28 under varying storage conditions. The
graphs provides the particle count (number of particles/mL)
determined for each of the listed equivalent circular diameter
ranges when stored at either 5.degree. C. or 30.degree. C. in
either a vial or a syringe. FIG. 80A provides the particle counts
within various ranges spanning 1 .mu.m to 100 .mu.m equivalent
circular diameter, and FIG. 80B provides the particle counts within
selected ranges spanning 10 .mu.m to 100 .mu.m equivalent circular
diameter. The legends from top to bottom correspond to the bars
from left to right for each particle diameter range.
[0095] FIGS. 81A and 81B show graphs depicting the subtracted MFI
results for Composition F29 under varying storage conditions. The
graphs provides the particle count (number of particles/mL)
determined for each of the listed equivalent circular diameter
ranges when stored at either 5.degree. C. or 30.degree. C. in
either a vial or a syringe. FIG. 81A provides the particle counts
within various ranges spanning 1 .mu.m to 100 .mu.m equivalent
circular diameter, and FIG. 81B provides the particle counts within
selected ranges spanning 10 .mu.m to 100 .mu.m equivalent circular
diameter. The legends from top to bottom correspond to the bars
from left to right for each particle diameter range.
[0096] FIGS. 82A and 82B show graphs depicting the subtracted MFI
results for Composition F30 under varying storage conditions. The
graphs provide the particle count (number of particles/mL)
determined for each of the listed equivalent circular diameter
ranges when stored at either 5.degree. C. or 30.degree. C. in
either a vial or a syringe. FIG. 82A provides the particle counts
within various ranges spanning 1 .mu.m to 100 .mu.m equivalent
circular diameter, and FIG. 82B provides the particle counts within
selected ranges spanning 10 .mu.m to 100 .mu.m equivalent circular
diameter. The legends from top to bottom correspond to the bars
from left to right for each particle diameter range.
[0097] FIGS. 83A and 83B show graphs depicting the subtracted MFI
results for Composition F31 under varying storage conditions. The
graphs provide the particle count (number of particles/mL)
determined for each of the listed equivalent circular diameter
ranges when stored at either 5.degree. C. or 30.degree. C. in
either a vial or a syringe. FIG. 83A provides the particle counts
within various ranges spanning 1 .mu.m to 100 .mu.m equivalent
circular diameter, and FIG. 83B provides the particle counts within
selected ranges spanning 10 .mu.m to 100 .mu.m equivalent circular
diameter. The legends from top to bottom correspond to the bars
from left to right for each particle diameter range.
[0098] FIGS. 84A and 84B show graphs comparing the subtracted MFI
results for Compositions F27 to F31 under varying storage
conditions. The graphs provide the particle count (number of
particles/mL) determined for each of the listed equivalent circular
diameter ranges when stored at either 5.degree. C. or 30.degree. C.
in either a vial or a syringe. FIG. 84A provides the particle
counts within various ranges spanning 1 .mu.m to 100 .mu.m
equivalent circular diameter, and FIG. 84B provides the particle
counts within selected ranges spanning 10 .mu.m to 75 .mu.m
equivalent circular diameter. In FIG. 84A, the particle count
within the range of <1 .mu.m to <2 .mu.m equivalent circular
diameter obtained for Composition F31 stored at 30.degree. C. in a
vial was 217,404, which exceeded the values depicted in the y-axis
of the graph, so this value is indicated above the corresponding
bar. In FIG. 84B, the particle count within the range of <10
.mu.m to <25 .mu.m equivalent circular diameter obtained for
Composition F31 stored at 30.degree. C. in a vial was 3,044, which
exceeded the values depicted in the y-axis of the graph, so this
value is indicated above the corresponding bar. The legends from
top to bottom correspond to the bars from left to right for each
particle diameter range.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and Abbreviations
[0099] As used herein, the following terms and phrases shall have
the meanings set forth herein.
[0100] The term "about" when used in connection with a referenced
numeric indication means the referenced numeric indication plus or
minus up to 10% of that referenced numeric indication. For example,
"about 100" means from 90 to 110.
[0101] The term "antagonist" refers to an agent that inhibits,
either partially or fully, the activity or production of a target
molecule. In particular, the term "antagonist," as applied
selectively herein, means an agent capable of decreasing levels of
gene expression, mRNA levels, protein levels or protein activity of
the target molecule. Illustrative forms of antagonists include, for
example, proteins, polypeptides, peptides (such as cyclic
peptides), antibodies or antibody fragments, peptide mimetics,
nucleic acid molecules, antisense molecules, ribozymes, aptamers,
RNAi molecules, and small organic molecules. Illustrative
non-limiting mechanisms of antagonist inhibition include repression
of one or both of ligand synthesis and stability (e.g., using,
antisense, ribozymes or RNAi compositions targeting the ligand
gene/nucleic acid), blocking of binding of the ligand to its
cognate receptor (e.g., using anti-ligand aptamers, antibodies,
anti-receptor antibodies, or a soluble, decoy cognate receptor or
fragment thereof), repression of one or both of receptor synthesis
and stability (e.g., using antisense, ribozymes or RNAi
compositions targeting the ligand receptor gene/nucleic acid),
blocking of the binding of the receptor to its cognate response
element (e.g., using anti-receptor antibodies) and blocking of the
activation of the receptor by its cognate ligand (e.g., using
receptor tyrosine kinase inhibitors). In addition, the antagonist
may directly or indirectly inhibit the target molecule.
[0102] As used herein, an "antibody" includes whole antibodies and
any antigen binding fragment or a single chain thereof. Thus the
term "antibody" includes any protein or peptide containing molecule
that comprises at least a portion of an immunoglobulin molecule
having biological activity of binding to the antigen. Examples of
such may comprise a complementarity determining region (CDR) of a
heavy or light chain or a ligand binding portion thereof, a heavy
chain or light chain variable region, a heavy chain or light chain
constant region, a framework (FR) region, or any portion thereof,
or at least one portion of a binding protein. Antibodies include
monoclonal antibodies and polyclonal antibodies.
[0103] The term "antibody fragment" includes a portion of an
antibody that is an antigen binding fragment or single chains
thereof. An antibody fragment can be a synthetically or genetically
engineered polypeptide. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include:
(i) a Fab fragment, a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains, (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region, (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains, (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341
544-546), which consists of a V.sub.H domain, and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, V.sub.L and V.sub.H, are coded for
by separate genes, they can be joined, using recombinant methods,
by a synthetic linker that enables them to be made as a single
protein chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (scFv), see e.g.,
Bird et al. (1988) Science 242 423-426, and Huston et al. (1988)
Proc Natl Acad Sci USA 85 5879-5883). Such single chain antibodies
are also intended to be encompassed within the term
"antigen-binding fragment" of an antibody. These antibody fragments
are obtained using conventional techniques known to those in the
art, and the fragments can be screened for utility in the same
manner as whole antibodies.
[0104] The term "aptamer" refers to a peptide or nucleic acid that
has an inhibitory effect on a target. Inhibition of the target by
the aptamer can occur by binding of the target, by catalytically
altering the target, by reacting with the target in a way which
modifies the target or the functional activity of the target, by
ionically or covalently attaching to the target as in a suicide
inhibitor or by facilitating the reaction between the target and
another molecule. Aptamers can be peptides, ribonucleotides,
deoxyribonucleotides, other nucleic acids or a mixture of the
different types of nucleic acids. Aptamers can comprise one or more
modified amino acid, bases, sugars, polyethylene glycol spacers or
phosphate backbone units as described in further detail herein.
Aptamers can be pegylated or unpegylated. For example, one or more
polyethylene glycol chains can be linked to the 5' end of a nucleic
acid aptamer via a linker.
[0105] A "composition" can comprise an active agent and a carrier,
inert or active. The compositions are useful for diagnostic or
therapeutic use in vitro, in vivo or ex vivo. In particular
embodiments, the compositions are sterile, substantially free of
endotoxins or non-toxic to recipients at the dosage or
concentration employed.
[0106] The term "label" includes, but is not limited to, a
radioactive isotope, a fluorophore, a chemiluminescent moiety, an
enzyme, an enzyme substrate, an enzyme cofactor, an enzyme
inhibitor, a dye, a metal ion, a ligand (e.g., biotin or a hapten)
and the like. Examples of fluorophore labels include fluorescein,
rhodamine, dansyl, umbelliferone, Texas red, and luminol. Other
examples of labels include NADPH, alpha-beta-galactosidase and
horseradish peroxidase.
[0107] The term "nucleic acid" refers to a polynucleotide such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term
also includes analogs of RNA or DNA made from nucleotide analogs,
and, as applicable to the embodiment being described, single (sense
or antisense) and double-stranded polynucleotides, expressed
sequence tags (ESTs), chromosomes, cDNAs, mRNAs, and rRNAs. Nucleic
acids include modified forms of nucleic acids that deviate
structurally from naturally occurring nucleic acid structures based
on the standard building blocks (adenosine, cytidine, guanosine,
thymidine and uridine). Modifications may be to the backbone, sugar
or nucleobase and can be naturally occurring or artificially
introduced. For example, nucleic acids may be modifed within their
backbone. Illustrative modifications are disclosed herein. Nucleic
acids can include nucleic acid aptamers and spiegelmers.
[0108] In some embodiments, Antagonist A exists in a modified form.
A modified form of Antagonist A is that which comprises a
nucleotide in a modified form as described herein, where the
nucleotide is present in an unmodified form in Antagonist A.
[0109] The terms "RNA interference," "RNAi," "miRNA," and "siRNA"
refer to any method by which expression of a gene or gene product
is decreased by introducing into a target cell one or more
double-stranded RNAs, which are homologous to a gene of interest
(particularly to the messenger RNA of the gene of interest, e.g.,
PDGF or VEGF).
[0110] The term "neovascularization" refers to new blood vessel
formation in abnormal tissue or in abnormal positions.
[0111] The term "angiogenesis" refers to formation of new blood
vessels in normal or in abnormal tissue or positions.
[0112] The term "ophthalmological disease" includes diseases of the
eye and diseases of the ocular adnexa.
[0113] The term "ocular neovascular disorder" refers to an ocular
disorder characterized by neovascularization. Certain cancers are
ocular neovascular disorders. In one embodiment, the ocular
neovascular disorder is a disorder other than cancer. Examples of
ocular neovascular disorders other than cancer include diabetic
retinopathy and age-related macular degeneration.
[0114] The term "mammal" includes human and non-human mammals, such
as, e.g., a human, mouse, rat, rabbit, monkey, cow, hog, sheep,
horse, dog, and cat.
[0115] The term "protein" and "polypeptide" are used
interchangeably and in their broadest sense refer to a compound of
two or more subunit amino acids, amino acid analogs or
peptidomimetics. The subunits may be linked by peptide bonds. In
another embodiment, the subunit may be linked by other bonds, e.g.,
ester, ether, etc. No limitation is placed on the maximum number of
amino acids which may comprise a protein's or peptide's
sequence.
[0116] As used herein the term "amino acid" refers to natural or
unnatural or synthetic amino acids, including glycine and both the
D and L optical isomers, amino acid analogs and
peptidomimetics.
[0117] The term "PDGF" refers to a platelet-derived growth factor
that regulates cell growth or division. As used herein, the term
"PDGF" includes the various subtypes of PDGF including PDGF-B
(e.g., GenBank Accession Nos. X02811 and CAa26579), PDGF-A (GenBank
Accession nos. X06374 and CAA29677), PDGF-C (GenBank Accession Nos.
NM 016205 and NP 057289), PDGF-D, variants 1 and 2 (GenBank
Accession Nos. NM 025208, NP 079484. NM 033135, NP 149126), and
dimerized forms thereof, including PDGF-AA, PDGF-AB, PDGF-BB,
PDGF-CC, and PDGF-DD. Platelet derived growth factors includes
homo- or heterodimers of A-chain (PDGF-A) and B-chain (PDGF-B) that
exert their action via binding to and dimerization of two related
receptor tyrosine kinase platelet-derived growth factor cell
surface receptors (i.e., PDGFRs), PDGFR-.alpha. (see GenBank
Accession Nos. NM 006206 and NP 006197) and PDGFR-.beta. (see
GenBank Accession Nos. NM 002609 and NP 002600). See, also, PCT
Application Publication No. 2010/127029, which is incorporated
herein in its entirety, for PDGF sequences. In addition, PDGF-C and
PDGF-D, two additional protease-activated ligands for the PDGFR
complexes, have been identified (Li et al., (2000) Nat. Cell. Biol.
2: 302-9; Bergsten et al. (2001) Nat. Cell. Biol. 3: 512-6; and
Uutele et al., (2001) Circulation 103: 2242-47). Due to the
different ligand binding specificities of the PDGFRs, it is known
that PDGFR-.alpha./.alpha. binds PDGF-AA, PDGF-BB. PDGF-AB, and
PDGF-CC; PDGFR-.beta./.beta. binds PDGF-BB and PDGF-DD; whereas
PDGFR-.alpha./.beta. binds PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD
(Betsholtz et al., (2001) BioEssavs 23: 494-507). As used herein,
the term "PDGF" also refers to those members of the class of growth
factors that induce DNA synthesis and mitogenesis through the
binding and activation of a PDGFR on a responsive cell type. PDGFs
can effect, for example: directed cell migration (chemotaxis) and
cell activation; phospholipase activation; increased
phosphatidylinositol turnover and prostaglandin metabolism;
stimulation of both collagen and collagenase synthesis by
responsive cells; alteration of cellular metabolic activities,
including matrix synthesis, cytokine production, and lipoprotein
uptake; induction, indirectly, of a proliferative response in cells
lacking PDGF receptors; and potent vasoconstrictor activity. The
term "PDGF" can be used to refer to a "PDGF" polypeptide, a "PDGF"
encoding gene or nucleic acid, or a dimerized form thereof. The
term "PDGF-A" refers to an A chain polypeptide of PDGF or its
corresponding encoding gene or nucleic acid. The term "PDGF-B"
refers to a B chain polypeptide of PDGF or its corresponding
encoding gene or nucleic acid. The term "PDGF-C" refers to a C
chain polypeptide of PDGF or its corresponding encoding gene or
nucleic acid. The term "PDGF-D" refers to a D chain polypeptide of
PDGF or its corresponding encoding gene or nucleic acid, including
variants 1 and 2 of the D chain polypeptide of PDGF. The term
"PDGF-AA" refers to a dimer having two PDGF-A chain polypeptides.
The term "PDGF-AB" refers to a dimer having one PDGF-A chain
polypeptide and one PDGF-B chain polypeptide. The term "PDGF-BB"
refers to a dimer having two PDGF-B chain polypeptides. The term
"PDGF-CC" refers to a dimer having two PDGF-C chain polypeptides.
The term "PDGF-DD" refers to a dimer having two PDGF-D chain
polypeptides.
[0118] The term "VEGF" refers to a vascular endothelial growth
factor that induces angiogenesis or an angiogenic process. As used
herein, the term "VEGF" includes the various subtypes of VEGF (also
known as vascular permeability factor (VPF) and VEGF-A) (see
GenBank Accesion Nos. NM 003376 and NP 003367) that arise by, e.g.,
alternative splicing of the VEGF-A/VPF gene including VEGF.sub.121,
VEGF.sub.165 and VEGF.sub.189. See, also, PCT Application
Publication No. 2010/127029, which is incorporated herein in its
entirety, for VEGF sequences. Further, as used herein, the term
"VEGF" includes VEGF-related angiogenic factors such as PIGF
(placenta growth factor), VEGF-B, VEGF-C, VEGF-D and VEGF-E, which
act through a cognate VEFG receptor (i.e., VEGFR) to induce
angiogenesis or an angiogenic process. The term "VEGF" includes any
member of the class of growth factors that binds to a VEGF receptor
such as VEGFR-I (FIt-I) (see GenBank Accession No. AF063657 and SID
NO:8 of PCT Application Publication No. WO 2010/127029), VEGFR-2
(KDR/Flk-1) (see GenBank Accession Nos. AF035121 and AAB88005), or
VEGFR-3 (FLT-4). The term "VEGF" can be used to refer to a "VEGF"
polypeptide or a "VEGF" encoding gene or nucleic acid.
[0119] The term "PDGF antagonist" refers generally to an agent that
reduces, or inhibits, either partially or fully, the activity or
production of a PDGF. A PDGF antagonist can directly or indirectly
reduce or inhibit the activity or production of a specific PDGF
such as PDGF-B. Furthermore. "PDGF antagonists," consistent with
the above definition of "antagonist," include agents that act on a
PDGF ligand or its cognate receptor so as to reduce or inhibit a
PDGF-associated receptor signal. Examples of "PDGF antagonists"
include antisense molecules, ribozymes or RNAi that target a PDGF
nucleic acid; anti-PDGF aptamers, anti-PDGF antibodies to PDGF
itself or its receptor, or soluble PDGF receptor decoys that
prevent binding of a PDGF to its cognate receptor; antisense
molecules, ribozymes or RNAi that target a cognate PDGF receptor
(PDGFR) nucleic acid; anti-PDGFR aptamers or anti-PDGFR antibodies
that bind to a cognate PDGFR receptor; and PDGFR tyrosine kinase
inhibitors.
[0120] The term "VEGF antagonist" refers generally to an agent that
reduces, or inhibits, either partially or fully, the activity or
production of a VEGF. A VEGF antagonist can directly or indirectly
reduce or inhibit the activity or production of a specific VEGF
such as VEGF.sub.165. Furthermore, "VEGF antagonists," consistent
with the above definition of "antagonist," include agents that act
on either a VEGF ligand or its cognate receptor so as to reduce or
inhibit a VEGF-associated receptor signal. Examples of "VEGF
antagonists" include antisense molecules, ribozymes or RNAi that
target a VEGF nucleic acid; anti-VEGF aptamers, anti-VEGF
antibodies to VEGF itself or its receptor, or soluble VEGF receptor
decoys that prevent binding of a VEGF to its cognate receptor;
antisense molecules, ribozymes, or RNAi that target a cognate VEGF
receptor (VEGFR) nucleic acid; anti-VEGFR aptamers or anti-VEGFR
antibodies that bind to a cognate VEGFR receptor, and VEGFR
tyrosine kinase inhibitors. As used herein, the term "VEGF
antagonist" is used to refer collectively to ranibizumab,
bevacizumab, and aflibercept.
[0121] "Pharmaceutically acceptable salts" include sulfate,
citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,
bisulfate, phosphate, acid phosphate, Isomcotinate, lactate,
salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,
bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,
gluconate, glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate,
trifluoroacetate, acrylate, chlorobenzoate, dimtrobenzoate,
hydroxybenzoate, methoxybcnzoate, methylbenzoate,
o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,
phenylbutyrate, alpha-hydroxybutyrate, butyne-1,4-dicarboxylate,
hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate,
glycollate, heptanoate, hippurate, malate, hydroxymaleate,
malonate, mandelate, mesylate, mcotinate, phthalate, teraphthalate,
propiolate, propionate, phenylpropionate, sebacate, suberate,
p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate,
2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate,
naphthalene-2-sulfonate, naphthalene-1,5-sulfonate,
xylenesulfonate, and tartarate salts. The term "pharmaceutically
acceptable salt" also refers to a salt of an antagonist of the
present invention having an acidic functional group, such as a
carboxylic acid functional group, and a base. Suitable bases
include, but are not limited to, hydroxides of alkali metals such
as sodium, potassium, and lithium, hydroxides of alkaline earth
metal such as calcium and magnesium, hydroxides of other metals,
such as aluminum and zinc, ammonia, and organic amines, such as
unsubstituted or hydroxy-substituted mono-, di-, or
tri-alkylamines, dicyclohexylamine, tributylamine, pyridine,
N-methyl, N-ethylamine, dicthylamine, triethylamine, mono-, bis-,
or tris-(2-OH-lower alkylamines), such as mono-, bis-, or
tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or
tris-(hydroxymethyl)methylamine, N,N-di-lower
alkyl-N-(hydroxyl-lower alkyl)-amines, such as
N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine,
N-methyl-D-glucamine, and amino acids such as arginine, lysine, and
the like. The term "pharmaceutically acceptable salt" also includes
a hydrate of a compound of the invention.
[0122] The term "effective amount," when used in connection with a
composition of the invention or treatment or prevention of an
ophthalmological disease, refers to a combined amount of a PDGF
antagonist and a VEGF antagonist that is useful to treat or prevent
an ophthalmological disease. The "effective amount" can vary
depending upon the mode of administration, specific locus of the
ophthalmological disease, the age, body weight, and general health
of the mammal. The effective amount of each antagonist of a
composition of the invention is the amount of each that is useful
for treating or preventing an ophthalmological disease with the
composition, even if the amount of the PDGF antagonist in the
absense of the VEGF antagonist, or the VEGF antagonist in the
absense of the PDGF antagonist, is ineffective to treat or prevent
the ophthalmological disease.
[0123] A "variant" of polypeptide X refers to a polypeptide having
the amino acid sequence of polypeptide X that is altered in one or
more amino acid residues. The variant can have "conservative"
changes, wherein a substituted amino acid has similar structural or
chemical properties (e.g., replacement of leucine with isoleucine).
More rarely, a variant can have "nonconservative" changes (e.g.,
replacement of glycine with tryptophan). Analogous minor variations
may also include amino acid deletions or insertions, or both.
Guidance in determining which amino acid residues may be
substituted, inserted, or deleted without eliminating biological or
immunological activity can be determined using computer programs
well known in the art, for example, LASERGENE software
(DNASTAR).
[0124] The term "variant," when used in the context of a
polynucleotide sequence, can encompass a polynucleotide sequence
related to that of a gene, coding sequence thereof, aptamer, or
other polynucleotide sequence. The variant may include one or more
nucleotide or nucleoside substitutions, additions or insertions as
compared to the reference gene, coding sequence, aptamer or other
polynucleotide sequence. This definition also includes, for
example, "allelic," "splice," "species," or "polymorphic" variants.
A splice variant can have significant identity to a reference
molecule, but will generally have a greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA
processing. Species variants are polynucleotide sequences that vary
from one species to another. A polymorphic variant is a variation
in the polynucleotide sequence of a particular gene between
individuals of a given species.
[0125] As used herein, the term "excipient" refers to a typically
inert substance that is commonly used as a diluent, vehicle,
preservative, binder, or stabilizing agent for active agents and
includes, but is not limited to, proteins (e.g., serum albumin,
etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine,
arginine, glycine, histidine, alanine, etc.), fatty acids and
phospholipids (e.g., alkyl sulfonates, caprylate, etc.),
surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.),
saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols
(e.g., mannitol, sorbitol, etc.). Also see Remington's
Pharmaceutical Sciences (by Joseph P. Remington, 18th ed., Mack
Publishing Co., Easton. Pa.) and Handbook of Pharmaceutical
Excipients (by Raymond C. Rowe, 5th ed., APhA Publications,
Washington, D.C.) which are hereby incorporated in its entirety. In
certain embodiments, the excipient(s) imparts a beneficial physical
property to the composition, such as increased protein,
polynucleotide, aptamer or small molecule stability, increased
protein, polynucleotide aptamer or small molecule solubility, or
decreased viscosity. In some embodiments, the compositions comprise
a plurality of active agents, and the excipient(s) help stabilize
the active agents.
[0126] The term "buffer" as used herein denotes a pharmaceutically
acceptable excipient, which stabilizes the pH of a pharmaceutical
preparation. Suitable buffers are well known in the art. Suitable
pharmaceutically acceptable buffers include but are not limited to
acetate-buffers, histidine-buffers, citrate-buffers,
succinate-buffers, tris-buffers and phosphate-buffers. Methods for
preparing such buffers are known in the art. Independently from the
buffer used, the pH can be adjusted at a value from about 4.5 to
about 7.0 or alternatively from about 5.5 to about 6.5 or
alternatively about 6.0 with an acid or a base known in the art,
e.g., succinic acid, hydrochloric acid, acetic acid, phosphoric
acid, sulfuric acid and citric acid, sodium hydroxide and potassium
hydroxide. Suitable buffers include, without limitation, histidine
buffer, 2-morpholinoethanesulfonic acid (MES), cacodylate,
phosphate, acetate, succinate, and citrate buffers. Additional
examples of phosphate buffers also include, without limitation,
sodium phosphate buffers and potassium phosphate buffers. Sodium
phosphate buffer may be prepared, e.g., by combining a solution of
NaH.sub.2PO.sub.4 (monobasic) with a solution of Na.sub.2HPO.sub.4
(dibasic) and then adjusting the pH of the combined solutions with
either phosphoric acid or sodium hydroxide to achieve the desired
pH. 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers may be
prepared, e.g., by adjusting the pH of a solution of Tris using HCl
to achieve a desired pH. e.g., a pH in the range of about pH 7.0 to
about pH 9.0. L-histidine may also be used as a buffer according to
the invention. In certain embodiments, a buffer is capable of
achieving or maintaining the pH of a composition of the invention
within a desired range or at or near a desired pH, e.g., during
storage. e.g., during storage at room temperature or 4.degree. C.
for at least one week, at least one month, at least two months, at
least four months, at least six months, at least one year, or at
least two years. In certain embodiments, the concentration of the
buffer is from about 0.01 mM to about 1000 mM, about 0.1 mM to
about 1000 mM, about 0.1 mM to about 500 mM, about 0.1 to about 200
mM, about 0.1 to about 100 mM, about 1 mM to about 1000 mM, about 1
mM to about 500 mM, about 1 mM to about 200 mM, about 1 mM to about
100 mM, about 1 mM to about 50 mM, about 2 mM to about 60 mM, about
4 mM to about 60 mM, or about 4 mM to about 40 mM, about 5 mM to
about 20 mM, or about 5 mM to about 25 mM.
[0127] Pharmaceutically acceptable "cryoprotectants" are known in
the art and include without limitation, e.g., sucrose, trehalose,
and glycerol. Pharmaceutically acceptable cryoprotectants provide
stability protection of compositions, or one or more active
ingredients therein, from the effects of freezing or
lyophilization.
[0128] The term "tonicity agent" or "tonicity modifier" as used
herein denotes pharmaceutically acceptable agents used to modulate
the tonicity of a composition. Suitable tonicity agents include,
but are not limited to, sodium chloride, sorbitol, trehalose,
potassium chloride, glycerin and any component from the group of
amino acids, sugars, as defined herein as well as combinations
thereof. In certain embodiments, tonicity agents may be used in an
amount of about 1 mM to about 1000 mM, about 1 mM to about 500 mM,
about 5 mM to about 500 mM, about 10 mM to about 450 mM, about 20
mM to about 400 mM, about 50 mM to about 300 mM, about 100 mM to
about 200 mM, or about 125 mM to about 175 mM. In certain
embodiments, a tonicity agent comprises an amino acid present in a
composition at about 5 mM to about 500 mM.
[0129] The term "stabilizer" indicates a pharmaceutical acceptable
excipient, which protects the active pharmaceutical ingredient(s)
or agents(s) or the composition from chemical or physical
degradation during manufacturing, storage and application.
Stabilizers include, but are not limited to, sugars, amino acids,
polyols, surfactants, antioxidants, preservatives, cyclodextrines,
e.g. hydroxypropyl-.beta.-cyclodextrine,
sulfobutylethyl-.beta.-cyclodextrin, .beta.-cyclodextrin,
polyethyleneglycols. e.g. PEG 3000, PEG 3350, PEG 4000, PEG 6000,
albumin, e.g. human serum albumin (HSA), bovine serum albumin
(BSA), salts, e.g. sodium chloride, magnesium chloride, calcium
chloride, and chelators, e.g. EDTA. Stabilizers may be present in
the composition in an amount of about 0.1 mM to about 1000 mM,
about 1 mM to about 500 mM, about 10 to about 300 mM, or about 100
mM to about 300 mM.
[0130] As used herein, the term "surfactant" refers to a
pharmaceutically acceptable organic substance having amphipathic
structures; namely, it is composed of groups of opposing solubility
tendencies, typically an oil-soluble hydrocarbon chain and a
water-soluble ionic group. Surfactants can be classified, depending
on the charge of the surface-active moiety, into anionic, cationic,
and nonionic surfactants. Surfactants may be used as wetting,
emulsifying, solubilizing, and dispersing agents for pharmaceutical
compositions and preparations of biological materials. In some
embodiments of the compositions described herein, the amount of
surfactant is described as a percentage expressed in weight/volume
percent (w/v %). Suitable pharmaceutically acceptable surfactants
include, but are not limited to, the group of
polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene
alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X),
polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic),
or sodium dodecyl sulphate (SDS). Polyoxyethylenesorbitan-fatty
acid esters include polysorbate 20, (sold under the trademark Tween
20.TM.) and polysorbate 80 (sold under the trademark Tween 80.TM.).
Polyethylene-polypropylene copolymers include those sold under the
names Pluronic.RTM. F68 or Poloxamer 188.TM.. Polyoxyethylene alkyl
ethers include those sold under the trademark Brij.TM..
Alkylphenolpolyoxyethylene ethers include those sold under the
tradename Triton-X. Polysorbate 20 (Tween 20.TM.) and polysorbate
80 (Tween 80.TM.) are generally used in a concentration range of
about 0.001% w/v to about 1% w/v or about 0.002% w/v to about 0.1%
w/v of the total volume of the composition, or alternatively of
about 0.003% w/v to about 0.007% w/v. In some embodiments, Tween
80.TM. is used at about 0.003% w/v, about 0.004% w/v, about 0.0045%
w/v, about 0.005% w/v, about 0.0055% w/v, about 0.006% w/v or about
0.007% w/v. In some embodiments, Tween 80.TM. is used at about
0.005% w/v. In this aspect, "w/v" intends the weight of surfactant
per total volume of the composition.
[0131] A "lyoprotectant" refers to a pharmaceutically acceptable
substance that stabilizes a protein, nucleic acid or other active
pharmaceutical ingredient(s) or agent(s) during lyophilization.
Examples of lyoprotectants include, without limitation, sucrose,
trehalose or mannitol.
[0132] A "polyol" refers to an alcohol containing multiple hydroxyl
groups, or a sugar alcohol. A sugar alcohol is a hydrogenated form
of carbohydrate, whose carbonyl group (aldehyde or ketone, reducing
sugar) has been reduced to a primary or secondary hydroxyl group
(hence the alcohol). Sugar alcohols have the general formula
H(HCHO).sub.n+1H, whereas sugars have H(HCHO).sub.nHCO.
[0133] An "antioxidant" refers to a molecule capable of slowing or
preventing the oxidation of other molecules. Antioxidants are often
reducing agents, chelating agents and oxygen scavengers such as
thiols, ascorbic acid or polyphenols. Non-limiting examples of
antioxidants include ascorbic acid (AA, E300), thiosulfate,
methionine, tocopherols (E306), propyl gallate (PG, E310), tertiary
butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA, E320) and
butylated hydroxytoluene (BHT, E321).
[0134] A "preservative" is a natural or synthetic chemical that is
added to products such as foods, pharmaceutical compositions,
paints, biological samples, wood, etc. to prevent decomposition by
microbial growth or by undesirable chemical changes. Preservative
additives can be used alone or in conjunction with other methods of
preservation. Preservatives may be antimicrobial preservatives,
which inhibit the growth of bacteria and fungi, or antioxidants
such as oxygen absorbers, which inhibit the oxidation of
constituents. Examples of antimicrobial preservatives include
benzalkonium chloride, benzoic acid, cholorohexidine, glycerin,
phenol, potassium sorbate, thimerosal, sulfites (sulfur dioxide,
sodium bisulfite, potassium hydrogen sulfite, etc.) and disodium
EDTA. Other preservatives include those commonly used in patenteral
protein compositions such as benzyl alcohol, phenol, m-cresol,
chlorobutanol or methylparaben.
[0135] The present invention provides compositions comprising at
least one anti-PDGF aptamer and at least one VEGF antagonist, as
well as related methods of manufacture and use thereof.
[0136] In one embodiment, the present invention provides a
composition comprising an effective amount of: (a) an anti-PDGF
aptamer or a pharmaceutically acceptable salt thereof; and (b) a
VEGF antagonist or a pharmaceutically acceptable salt thereof. In
particular embodiments, at least about 90% of one or both of the
anti-PDGF aptamer and the VEGF antagonist is chemically stable when
the composition is stored at a temperature from about 2.0.degree.
C. to about 8.0.degree. C. for at least about twelve weeks.
[0137] In particular embodiments of various compositions and
methods of the present invention, the anti-PDGF aptamer is
Antagonist A or a modified form thereof. In particular embodiments
of various compositions and methods of the present invention, the
VEGF antagonist is ranibizumab, bevacizumab, or aflibercept, or
pharmaceutically acceptable salts thereof.
[0138] In another embodiment, the present invention provides
methods for treating or preventing an ophthalmological disease,
comprising administering to a mammal in need thereof a composition
of the invention. The composition is administered in an amount
effective to treat or prevent the ophthalmological disease. In
various embodiments, the ophthalmological disease is age-related
macular degeneration, polypoidal choroidal vasculopathy, condition
associated with choroidal neovascularization, hypertensive
retinopathy, diabetic retinopathy, sickle cell retinopathy,
condition associated with peripheral retinal neovascularization,
retinopathy of prematurity, venous occlusive disease, arterial
occlusive disease, central serous chorioretinopathy, cystoid
macular edema, retinal telangiectasia, arterial macroaneurysm,
retinal angiomatosis, radiation-induced retinopathy, rubeosis
iridis, or a neoplasm. In particular embodiments, the
ophthalmological disease is age-related macular degeneration, and
the age-related macular degeneration is wet age-related macular
degeneration or dry age-related macular degeneration. In certain
embodiments, the composition is present in a drug-delivery device.
In certain embodiments, the composition is administered
intraocularly. In specific embodiments, the intraocular
administration is intravitreal administration or anterior chamber
administration. In other embodiments, the mammal is a human.
PDGF Aptamers and VEGF Antagonists
[0139] The present invention provides compositions, including
pharmaceutical compositions, comprising an anti-PDGF aptamer and a
VEGF antagonist. In particular embodiments, the anti-PDGF aptamer
is Antagonist A or a modified form thereof (or a pharmaceutically
acceptable salt thereof), and the VEGF antagonist is ranibizumab,
bevacizumab, or aflibercept (or a pharmaceutically acceptable salt
thereof). The present invention further provides compositions
comprising an effective amount of an anti-PDGF aptamer and a VEGF
antagonist.
[0140] Anti-PDGF Aptamers
[0141] In certain embodiments, anti-PDGF aptamers include, but are
not limited to, those described in U.S. Pat. No. 8,039,443,
incorporated by reference herein in its entirety, which include
both PDGF-specific and PDGF-VEGF-specific aptamers. Examples of
anti-PDGF aptamers include aptamers whose oligonucleotide sequence
comprises, consists essentially of or consists of one of the
following sequences: ARC126:
5'-(5'-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG-3'. SEQ ID NO:
1)-HEG-(5'-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3', SEQ ID
NO:2)-HEG-(5'-T-dG-dA-T-fC-fC-fU-mG-3'dT-3', SEQ ID NO:3)-3'
wherein HEG=hexacthylene glycol amidite; ARC127: 5'-[40K
PEG]-(5'-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG-3', SEQ ID NO:
1)-HEG=(5'-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3', SEQ ID
NO:2)-HEG-(5'-T-dG-dA-T-fC-fC-fU-mG-3'dT-3', SEQ ID NO:3)-3'
wherein HEG=hexaethylene glycol amidite; ARC240: 5'-[20K
PEG]-(5'-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG-3', SEQ ID
NO:1)-HEG-(5'-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3', SEQ ID
NO:2)-HEG-(5'-T-dG-dA-T-fC-fC-fU-mG-3'dT-3', SEQ ID NO:3)-3'
wherein HEG=hexaethylene glycol amidite; ARC308: 5'-[30K
PEG]-(5'-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG-3'. SEQ ID
NO:1)-HEG-(5'-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3', SEQ ID
NO:2)-HEG-(5'-T-dG-dA-T-fC-fC-fU-mG-3'dT-3', SEQ ID NO:3)-3'
wherein HEG=hexaethylene glycol amidite; deoxyARC126:
5'-dCdAdGdGdCdTdAdCdGdCdGdTdAdGdAdGdCdAdTdCdAdTdGdAdTdCdCdTdG-[3T]-3'
(SEQ ID NO:75) wherein "d" indicates unmodified deoxynucleotides
and "[3T]" refers to an inverted thymidine nucleotide that is
attached to the 3' end of the oligonucleotide at the 3' position on
the ribose sugar, thus the oligonucleotide has two 5' ends and is
thus resistant to nucleases acting on the 3' hydroxyl end; and
ARC24:
TABLE-US-00001 (SEQ ID NO: 6)
5'CACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTG 3'InvdT.
[0142] Examples of PDGF/VEGF binding multivalent aptamers include
the PDGF-B-VEGF aptamer chimeras TK.131.12. A and TK.131.12.B,
which allow for the simultaneous targeting of PDGF-B and VEGF.
These aptamera chimeras are described in PCT Patent Application
Publication Nos. WO2006/050498 and WO2004/094164. The sequence of
TK.131.012.A is:
5'dCdAdGdGdCdTdAdCdGmAmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCm
GmCmGmCmAmUdCdGdTdAdGdAdGdCdAdTdCdAdGdAdAdAdTdGdAdTdCdCdTdG[3 T]-3'
(SEQ ID NO:4), wherein "m" indicates 2'-OMe nucleotides. "d" and
"[3T]" are as defined above; and the sequence of TK.131.012.B is:
5'dCdAdGdGdCdTdAdCdGmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGm
CmGmCmAdCdGdTdAdGdAdGdCdAdTdCdAdGdAdAdAdTdGdAdTdCdCdTdG-[3T](SEQ ID
NO:5) wherein "m" and "[3T]" are as defined above.
[0143] In particular embodiments, an anti-PDGF aptamer binds PDGF.
Examples of anti-PDGF aptamers include a series of nucleic acid
aptamers of 31-35 nucleotides in length (SEQ ID NO: 1 to SEQ ID
NO:3, SEQ ID NO:4 to SEQ ID NO:30, SEQ ID NO:31 to SEQ ID NO:68,
SEQ ID NO:69, and SEQ ID NO:70 to SEQ ID NO:74 of U.S. Pat. No.
8,039,443), that bind specifically to PDGF-B protein in vitro and
which functionally block the activity of PDGF-BB in vivo and
cell-based assays. In particular embodiments, the anti-PDGF-B
aptamers are derived from a parent molecule ARC126 (5'-(SEQ ID
NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3'-dT-3') which contains
seven individual 2'F containing residues. The 2'F containing
residues can increase the in vitro serum and in vivo stability of
the aptamer by blocking its degradation by serum endonucleases or
exonucleases. In particular embodiments, the anti-PDGF aptamers are
fully 2'F-free aptamers that retain potent in vitro binding and
anti-proliferative activity and contain naturally occurring 2'deoxy
or 2'OMe substituted nucleotides. In addition, in particular
embodiments, these aptamers retain substantial serum stability as
determined through resistance to nuclease degradation in an in
vitro stability assay.
[0144] In certain embodiments, the anti-PDGF aptamer is Antagonist
A or a pharmaceutically acceptable salt thereof. The chemical name
of Antagonist A is [(monomethoxy 20K polyethylene glycol
carbamoyl-N2-) (monomethoxy 20K polyethylene glycol
carbamoyl-N6-)]-lysine-amido-6-hexandilyl-(1-5')-2'-deoxycytidylyl-(3'-5'-
)-2'-deoxyadenylyl-(3'-5')-2'-deoxyguanylyl-(3'-5')-2'-deoxyguanylyl-(3'-5-
')-2'-deoxycytidylyl-(3'-5')-2'-deoxy-2'-fluorouridylyl-(3'-5')-2'-deoxyad-
enylyl-(3'-5')-2'-deoxy-2'-fluorocytidylyl-(3'-5')-2'-deoxy-2'-methoxyguan-
ylyl-(3'-1)-PO.sub.3-hexa(ethyloxy)-(18-5')-2'-deoxycytidylyl-(3'-5')-2'-d-
eoxyguanylyl-(3'-5')-thymidylyl-(3'-5')-2'-deoxyadenylyl-(3'-5')-2'-deoxy--
2'-methoxyguanylyl-(3'-5')-2'-deoxyadenylyl-(3'-5')-2'-deoxy-2'-methoxygua-
nylyl-(3'-5')-2'-deoxycytidylyl-(3'-5')-2'-deoxyadenylyl-(3'-5')-2'-deoxy--
2'-fluorouridylyl-(3'-5')-2'-deoxy-2'-fluorocytidylyl-(3'-5')-2'-deoxy-2'--
methoxyadenylyl-(3'-1)-PO.sub.3-hexa(ethyloxy)-(18-5')-thymidylyl-(3'-5')--
2'-deoxyguanylyl-(3'-5')-2'-deoxyadenylyl-(3'-5')-thymidylyl-(3'-5')-2'-de-
oxy-2'-fluorocytidylyl-(3'-5')-2'-deoxy-2'-fluorocytidylyl-(3'-5')-2'-deox-
y-2'-fluorouridylyl-(3'-5')-2'-methoxyguanylyl-(3'-3')-thymidine.
[0145] The structure of Antagonist A is shown in FIGS. 78A-F, and
it is also described in FIG. 7 of PCT Application Publication No.
WO 2010/127029, which is incorporated herein its entirety.
[0146] The sequence of Antagonist A is:
TABLE-US-00002 (SEQ ID Nos. 1-3) 5'-[mPEG2 40
kD]-[HN-(CH.sub.2).sub.6O]CAGGCU.sub.fAC.sub.fG.sub.m[PO.sub.3(CH.sub.2CH-
.sub.2
O).sub.6]CGTAG.sub.mAG.sub.mCAU.sub.fC.sub.fA.sub.m[PO.sub.3(CH.sub.2CH.su-
b.2O).sub.6]TGATC.sub.fC.sub.fU.sub.fG.sub.m-iT-3',
where
[0147] [mPEG2 40 kD] represents two 20 kD polyethylene glycol (PEG)
polymer chains, in one embodiment two about 20 kD PEG polymer
chains, that are covalently attached to the two amino groups of a
lysine residue via carbamate linkages. This moiety is in turn
linked with the oligonucleotide via the amino linker described
below.
[0148] [HN-(CH.sub.2).sub.6O] represents a bifunctional
.alpha.-hydroxy-.omega.-amino linker that is covalently attached to
the PEG polymer via an amide bond. The linker is attached to the
oligonucleotide at the 5'-end of Antagonist A by a phosphodiester
linkage.
[0149] [PO.sub.3(CH.sub.2CH.sub.2O).sub.6] represents the
hexaethylene glycol (HEX) moieties that join segments of the
oligonucleotide via phosphodiester linkages. Antagonist A has two
HEX linkages that join together the 9.sup.th and 10.sup.th
nucleotides and 21.sup.st and 22.sup.nd nucleotides via
phosphodiester linkages between the linker and the respective
nucleotides.
[0150] C, A, G, and T represent the single letter code for the
2'-deoxy derivatives of cytosine, adenosine, guanosine, and
thymidine nucleic acids, respectively. Antagonist A has four
T-deoxyribocytosine, six T-deoxyriboadenosine, four
2'-deoxyriboguanosine, and four 2'-deoxyribothymidine.
[0151] G.sub.m and A.sub.m represent 2'-methoxy substituted forms
of guanosine and adenosine, respectively. Antagonist A has four
2'-methoxyguanosines and one 2'-methoxyadenosine. C.sub.f and
U.sub.f represent the 2'-fluoro substituted forms of cytosine and
uridine, respectively. Antagonist A has four 2'-fluorocytosines and
three 2'-fluorouridines.
[0152] The phosphodiester linkages in the oligonucleotide, with the
exception of the 3'-terminus, connect the 5'- and 3'-oxygens of the
ribose ring with standard nucleoside or nucleotide phosphodiester
linkages. The phosphodiester linkage between the 3'-terminal
thymidine and the penultimate G.sub.m links their respective
3'-oxygens, which is referred to as the 3',3'-cap.
[0153] Antagonist A has a molecular weight from about 40,000 to
about 60,000 Daltons for the entire molecule (including the nucleic
acid, amino linker and polyethylene glycol moieties), in one
embodiment from 40,000 to 60,000 Daltons, and can be colorless to
slightly yellow in solution. Antagonist A can be present in a
solution of monobasic sodium phosphate monohydrate and dibasic
sodium phosphate heptahydrate as buffering agents and sodium
chloride as a tonicity adjuster. Antagonist A is a hydrophilic
polymer. The Antagonist A sodium salt is soluble in water and in
phosphate-buffered saline (PBS), as assessed by visual inspection,
to at least about 50 mg (based on oligonucleotide weighty) mL
solution.
[0154] In one embodiment, Antagonist A is manufactured using an
iterative chemical synthesis procedure to produce the
oligonucleotide portion and amino linker, which is then covalently
bonded to a pegylation reagent, as shown in Example 4 and as
described in Example 4 of PCT Application Publication No. WO
2010/127029, which is hereby incorporated by reference in its
entirety.
[0155] Antagonist A can possess a sufficiently basic functional
group, which can react with any of a number of inorganic and
organic acids, to form a pharmaceutically acceptable salt. A
pharmaceutically-acceptable acid addition salt is formed from a
pharmaceutically-acceptable acid, as is well known in the art. Such
salts include those described herein and the pharmaceutically
acceptable salts listed in Journal of Pharmaceutical Science, 66,
2-19 (1977) and The Handbook of Pharmaceutical Salts, Properties,
Selection, and Use, P H Stahl and C G Wermuth (ED s), Verlag,
Zurich (Switzerland) 2002, which are hereby incorporated by
reference in their entirety.
[0156] In other embodiments, the anti-PDGF aptamer is a modified
form of an aptamer, such as Antagonist A, or another aptamer
described herein, which may include one or more of the
modifications described herein. Although discussed specifically
with respect to Antagonist A, it is understood that any of the
modifications described herein may be present in a modified form of
any other anti-PDGF aptamer described herein, each of which may be
useful in the present invention. In particular embodiments, a
modified form of an aptamer, e.g., a modified form of Antagonist A,
comprises or consists of the same nucleotide sequence and nucleic
acids as the aptamer, but comprises one or more different
polyethylene glycol polymer chains as compared to the aptamer, or
comprises one or more different linkers coupling one or more of the
polyethylene glycol polymer chains to the nucleic acid portion of
the aptamer.
[0157] In some embodiments, a modified form of an aptamer, e.g., a
modified form of Antagonist A, can have chemically modified
nucleotides as compared to the aptamer, including 5-X or 2'-Y
substitutions in pyrimidine bases and 8-X or 2'-Y substitutions in
purine bases. 2'-Modifications, such as 2'-fluoro and 2'-O-Me, can
be utilized for stabilization against nucleases without
compromising the aptamer binding interaction with the target. See,
e.g., Lin et al., Nucleic Acids Res., 22, 5229-5234 (1994);
Jellinek et al., Biochemistry, 34, 11363-1137 (1995); Lin et al.,
Nucleic Acids Res., 22, 5229-5234 (1994); Kubik et al., J.
Immunol., 159(1), 259-267 (1997); Pagratis et al., Nat.
Biotechnol., 1, 68-73 (1997); and Wilson et al., Curr Opin Chem
Biol, 10(6), 607-614 (2006). In some embodiments, the chemical
substitution can be a chemical substitution at a sugar position, a
chemical substitution at a base position, or a chemical
substitution at a phosphate position.
[0158] Modifications that may be present in modified forms of an
aptamer, e.g., Antagonist A, include, but are not limited to, those
which provide other chemical groups that incorporate additional
charge, polarizability, hydrophobicity, hydrogen bonding,
electrostatic interaction, or fluxionality to the aptamer bases or
to the aptamer as a whole. Such modifications include, but are not
limited to, 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
exocyclic amines, substitution of 4-thiouridine, substitution of
5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate
or alkyl phosphate modifications, methylations, unusual
base-pairing combinations such as the isobases isocytidine and
isoguanidine and the like. Modifications can also include 3' and 5'
modifications such as capping or modification with sugar moieties.
In some embodiments of the invention, the modified forms of an
aptamer, e.g., modified forms of Antagonist A, are RNA molecules
that are 2'-fluoro (2'-F) modified on the sugar moiety of
pyrimidine residues. Examples of modifications that may be present
in modified forms of an aptamer, e.g., modified forms of Antagonist
A, as well as stabilized aptamers that may be used according to the
present invention, are described in U.S. Pat. No. 8,039,443, which
is hereby incorporated by reference in its entirety. In certain
embodiments, the anti-PDGF aptamer is an anti-PDGF-B aptamer,
including but not limited to those described in U.S. Pat. No.
8,039,443.
[0159] In some embodiments, the stability of the aptamer can be
increased by the introduction of such modifications and as well as
by modifications and substitutions along the phosphate backbone of
the RNA, which may also be present in modified forms of the
aptamer, e.g., modified forms of Antagonist A. In addition, a
variety of modifications can be made on the nucleobases themselves
which both inhibit degradation and which can increase desired
nucleotide interactions or decrease undesired nucleotide
interactions. Accordingly, once the sequence of an aptamer is
known, modifications or substitutions can be made by the synthetic
procedures described below or by procedures known to those of skill
in the art. Any such modifications may be present in a modified
form of Antagonist A.
[0160] Other modifications that may be present in a modified form
of an aptamer, e.g., modified form of Antagonist A, include the
incorporation of modified bases (or modified nucleoside or modified
nucleotides) that are variations of standard bases, sugars or
phosphate backbone chemical structures occurring in ribonucleic
(i.e., A, C, G and U) and deoxyribonucleic (i.e., A, C, G and T)
acids. Included within this scope are, for example: Gm
(2'-methoxyguanylic acid), Am (2'-methoxyadenylic acid). Cf
(2'-fluorocytidylic acid), Uf (2'-fluorouridylic acid), Ar
(riboadenylic acid). A modified form of Antagonist A can include
cytosine or any cytosine-related base including 5-methylcytosine,
4-acetylcytosine, 3-methylcytosine, 5-hydroxymethyl cytosine,
2-thiocytosine, 5-halocytosine (e.g., 5-fluorocytosine,
5-bromocytosine, 5-chlorocytosine, and 5-iodocytosine), 5-propynyl
cytosine, 6-azocytosine, 5-trifluoromethylcytosine,
N4,N4-ethanocytosine, phenoxazine cytidine, phenothiazine cytidine,
carbazole cytidine or pyridoindole cytidine. A modified form of
Antagonist A can include guanine or any guanine-related base
including 6-methylguanine, 1-methylguanine, 2,2-dimethylguanine,
2-methylguanine, 7-methylguanine, 2-propylguanine, 6-propylguanine,
8-haloguanine (e.g., 8-fluoroguanine, 8-bromoguanine,
8-chloroguanine, and 8-iodoguanine), 8-aminoguanine,
8-sulfhydrylguanine, 8-thioalkylguanine, 8-hydroxylguanine,
7-methylguanine, 8-azaguanine, 7-deazaguanine or 3-deazaguanine. A
modified form of an aptamer, e.g., a modified form of Antagonist A,
may include adenine or any adenine-related base including
6-methyladenine, N6-isopentenyladenine. N6-methyladenine,
1-methyladenine, 2-methyladenine,
2-methylthio-N6-isopentenyladenine, 8-haloadenine (e.g.,
8-fluoroadenine, 8-bromoadenine, 8-chloroadenine, and
8-iodoadenine), 8-aminoadenine, 8-sulfhydryladenine,
8-thioalkyladenine, 8-hydroxyladenine, 7-methyladenine,
2-haloadenine (e.g., 2-fluoroadenine, 2-bromoadenine,
2-chloroadenine, and 2-iodoadenine), 2-aminoadenine, 8-azaadenine,
7-deazaadenine or 3-deazaadenine. Also included are uracil or any
uracil-related base including 5-halouracil (e.g., 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil),
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil,
1-methylpseudouracil, 5-methoxyaminomethyl-2-thiouracil,
5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methylaminomethyluracil,
5-propynyl uracil, 6-azouracil, or 4-thiouracil.
[0161] Examples of other modified base variants known in the art,
which may be present in a modified version of an aptamer, e.g., a
modified version of Antagonist A, include, without limitation,
4-acetylcytidine, 5-(carboxyhydroxylmethyl) uridine,
2'-methoxycytidine, 5-carboxymethylaminomethyl-2-thioridine,
5-carboxymethylaminomethyluridine, dihydrouridine,
2'-O-methylpseudouridine, b-D-galactosylqueosine, inosine,
N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine,
1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine,
2-methyladenosine, 2-methylguanosine, 3-methylcytidine,
5-methylcytidine, N6-methyladenosine, 7-methylguanosine,
5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,
b-D-mannosylqueosine, 5-methoxycarbonylmethyluridine,
5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine,
N-((9-b-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine,
N-((9-b-D-ribofuranosylpurine-6-yl)N-methyl-carbamoyl)threonine,
urdine-5-oxyacetic acid methylester, uridine-5-oxy acetic acid,
wybutoxosine, pseudouridine, queosine, 2-thiocytidine,
5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine,
5-methyluridine,
N-((9-b-D-ribofuranosylpurine-6-yl)carbamoyl)threonine,
2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine,
3-(3-amino-3-carboxypropyl)uridine.
[0162] Examples of modified nucleoside and nucleotide sugar
backbone variants known in the art include, without limitation,
those having, e.g. 2'-ribosyl substituents such as F, SH,
SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2, CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2ON(CH.sub.3).sub.2,
OCH.sub.2OCH.sub.2N(CH.sub.3).sub.2, O(C.sub.1-10 alkyl),
O(C.sub.2-10 alkenyl), O(C.sub.2-10 alkynyl), S(C.sub.1-10 alkyl),
S(C.sub.2-10 alkenyl), S(C.sub.2-10 alkynyl), NH(C.sub.1-10 alkyl),
NH(C.sub.2-10 alkenyl), NH(C.sub.2-10 alkynyl), and
O-alkyl-O-alkyl. Desirable 2' ribosyl substituents include
2'-methoxy (2'-OCH.sub.3), 2'-aminopropoxy (2'
OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2), 2'-amino (2'-NH.sub.2), and
2'-fluoro (2'-F). The 2'-substituent may be in the arabino (up)
position or ribo (down) position. These may be present in a
modified form of Antagonist A.
[0163] Examples of modifications include: a purine substitution for
a pyrimidine; a 2'-deoxy dihydrouridine substitution for a uridine;
a 2'-deoxy-5-methyl cytidine for a cytidine; a 2-amino purine
substitution for a purine; a phosphorothioate substituted for a
phosphodiester; a phosphorodithioate substituted for a
phosphodiester, a deoxynucleotide substituted for a 2'-OH
nucleotide; a 2'-OMe nucleotide, a 2'-fluoro nucleotide or a
2'-O-methoxyethyl nucleotide substituted for a 2'-OH or
deoxynucleotide; the addition of a PEG or PAG polymer; the addition
of a large steric molecule; the addition of a 3' cap; or any other
modification known to block nuclease degradation. See, for example,
U.S. Patent Publication No. 20090075342, which is incorporated by
reference in its entirety.
[0164] Modified forms of an aptamer. e.g., modified forms of
Antagonist A, may be made up of nucleotides or nucleotide analogs
such as described herein, or a combination of both, or are
oligonucleotide analogs. Modified forms of an aptamer, e.g.,
modified forms of Antagonist A, may contain nucleotide analogs at
positions which do not affect the function of the oligomer, for
example, to bind PDGF.
[0165] The anti-PDGF aptamers described herein can be linked with
one or more non-physiologically active groups, such as a lipophilic
compound (e.g., cholesterol); linked with one or more
non-immunogenic high molecular weight compounds (e.g., polyalkylene
glycol); or attached to or encapsulated in a complex comprising a
lipophilic component (e.g., a liposome). In one embodiment, the
linked aptamers enhance the cellular uptake of the aptamers by a
cell for delivery of the aptamers to an intracellular target. U.S.
Pat. No. 6,011,020, incorporated by reference herein in its
entirety, describes a method for preparing aptamers linked with one
or more lipophilic compounds or non-immunogenic, high molecular
weight compounds.
[0166] The anti-PDGF aptamers described herein may be attached via
a linker to one or more non-physiologically active groups, such as
lipophilic or Non-immunogenic, High Molecular Weight compounds, in
a diagnostic or therapeutic complex as described in U.S. Pat. No.
6,011,020. Aptamers that are attached via a linker to a Lipophilic
Compound, such as diacyl glycerol or dialkyl glycerol, in a
diagnostic or therapeutic complex are described in U.S. Pat. No.
5,859,228. Aptamers that are attached via a linker to a Lipophilic
Compound, such as a glycerol lipid, or to a Non-immunogenic, High
Molecular Weight Compound, such as polyalkylene glycol, are further
described in U.S. Pat. No. 6,051,698. Aptamers that are attached
via a linker to a Non-immunogenic, High Molecular Weight compound
or to a lipophilic compound are also further described in
PCT/US97/18944, filed Oct. 17, 1997, entitled "Vascular Endothelial
Growth Factor (VEGF) Nucleic Acid Ligand Complexes." Each of the
herein described patents and patent applications are specifically
incorporated by reference herein in its entirety.
[0167] One or more aptamers. e.g., Antagonist A, may be attached
via a linker to a Non-Immunogenic, High Molecular Weight compound
or lipophilic compound. A Non-Immunogenic, High Molecular Weight
compound can be a linear or branched compound that has a molecular
weight of about 100 Da to 1,000,000 Da, about 1000 Da to 500,000
Da, or about 1000 Da to 200,000 Da, that typically does not
generate an immunogenic response. In one embodiment, the
Non-Immunogenic. High Molecular Weight compound can be a
polyalkylene glycol. In one embodiment, the Non-Immunogenic, High
Molecular Weight compound comprises a polyalkylene glycol. In one
embodiment, the Non-Immunogenic. High Molecular Weight compound
comprises a plurality of polyalkylene glycols. In one embodiment,
the Non-Immunogenic, High Molecular Weight compound comprises two
polyalkylene glycols. In another embodiment, the polyalkylene
glycol can be polyethylene glycol (PEG). In some embodiments, the
PEG has a molecular weight of about 10 to about 80 kDa or a
molecular weight of about 20 to about 45 kDa. In some embodiments,
the plurality of PEGs has a combined molecular weight of about 10
to about 80 kDa or a molecular weight of about 20 to about 45 kDa.
In other embodiments, the Non-immunogenic, High Molecular Weight
compound comprises two polyalkylene glycols, each of which has a
molecular weight of about 20 kDa.
[0168] An aptamer, e.g., Antagonist A, may be attached via a linker
to one or more lipophilic compounds. Lipophilic compounds are
compounds that have the propensity to associate with or partition
into lipid or other materials or phases having a low dielectric
constant, including compounds based mostly on lipophilic
components. Lipophilic compounds include lipids as well as
non-lipid containing compounds that have the propensity to
associate with lipids (or other materials or phases with low
dielectric constants). Cholesterol, phospholipid, and glycerol
lipids, such as dialkyl glycerol, diacyl glycerol, and glycerol
amide lipids are further examples of lipophilic compounds. In one
embodiment, the lipophilic compound is a glycerol lipid.
[0169] The Non-immunogenic, High Molecular Weight compound or
lipophilic compound can be covalently bound via a linker to a
variety of positions on the aptamer, such as to an exocyclic amino
group on a nucleotide's base, the 5-position of a pyrimidine
nucleotide, the 8-position of a purine nucleotide, the hydroxyl
group of a nucleotide's phosphate, or a hydroxyl group or other
group at the 5' or 3' terminus of the aptamer. In some embodiments
where the lipophilic compound is a glycerol lipid, or the
Non-Immunogenic, High Molecular Weight compound is polyalkylene
glycol or polyethylene glycol, the Non-Immunogenic, High Molecular
Weight compound can be bonded via a linker to the 5' or 3' hydroxyl
of the phosphate group thereof. In one embodiment, the lipophilic
compound or Non-Immunogenic. High Molecular Weight compound is
bonded via a linker to the 5' phosphate group of the aptamer.
Attachment of the Non-Immunogenic, High Molecular Weight compound
or lipophilic compound to the aptamer can be done directly or with
the utilization of one or more linkers that interpose between the
aptamer and lipophilic compound or Non-Immunogenic, High Molecular
Weight compound. When attachment is done directly, in some
embodiments, no linker is present.
[0170] A linker is a molecular entity that connects two or more
molecular entities through covalent bonds or non-covalent
interactions, and can allow spatial separation of the molecular
entities in a manner that preserves the functional properties of
one or more of the molecular entities.
[0171] In one embodiment of the invention, the Non-Immunogenic,
High Molecular Weight Compound is a polyalkylene glycol and has the
structure R(O(CH.sub.2).sub.x).sub.nO--, where R is independently H
or CH.sub.3, x=2-5, and n.about.MW of the Polyalkylene
Glycol/(16+14x). In one embodiment of the present invention, the
molecular weight of the Polyalkylene Glycol is about between 10-80
kDa. In another embodiment, the molecular weight of the
Polyalkylene Glycol is about between 20-45 kDa. In yet another
embodiment, x=2 and n=9.times.10.sup.2. There can be one or more
Polyalkylene Glycols attached via a linker to the same aptamer. In
one embodiment, a plurality of Polyalkylene Glycols is attached via
a linker to the same aptamer. In another embodiment, two
Polyalkylene Glycols are attached via a linker to the same aptamer.
In another embodiment, Polyalkylene Glycols is a polyethylene
glycol that has a molecular weight of about 40 kDa.
[0172] In one embodiment, an anti-PDGF aptamer is attached via a
linker to a Non-Immunogenic, High Molecular Weight Compound such as
Polyalkylene Glycol or PEG, or to a plurality of Non-Immunogenic,
High Molecular Weight Compounds. In this embodiment, the
pharmacokinetic properties of the linked PDGF aptamer are improved
relative to the anti-PDGF aptamer alone. The Polyalkylene Glycol or
PEG can be covalently bound via a linker to a variety of positions
on the PDGF aptamer. In embodiments where Polyalkylene Glycol or
PEG are used, the anti-PDGF aptamer can be bonded via a linker
through the 5' hydroxyl group via a phosphodiester linkage.
[0173] In some embodiments, a plurality of aptamers can be
associated with a single Non-Immunogenic. High Molecular Weight
Compound, such as Polyalkylene Glycol or PEG, or a Lipophilic
Compound, such as a glycerolipid. The aptamers can all be to one
target or to different targets. In embodiments where a compound
comprises more than one anti-PDGF aptamer, there can be an increase
in avidity due to multiple binding interactions with the target,
PDGF. In yet further embodiments, a plurality of one or more of
Polyalkylene Glycol, PEG, and glycerol lipid molecules can be
attached to each other, to the same linker, or to a plurality of
linkers. In these embodiments, one or more aptamers can be
associated with each Polyalkylene Glycol, PEG, or glycerol lipid.
This can result in an increase in avidity of each aptamer to its
target. In addition, in embodiments where there are aptamers to
PDGF or aptamers to PDGF and different targets associated with
Polyalkylene Glycol, PEG, or glycerol lipid, a drug can also be
associated with, e.g., covalently bonded to, Polyalkylene Glycol,
PEG, or glycerol lipid. Thus the compound would provide targeted
delivery of the drug, with Polyalkylene Glycol. PEG, or glycerol
lipid serving as a linker, optionally, with one or more additional
linkers.
[0174] In particular embodiments, aptamers can be 5'-capped and/or
3'-capped with a 5'-5' inverted nucleotide cap structure at the 5'
end and/or a 3'-3' inverted nucleotide cap structure at the 3' end.
In certain embodiments, Antagonist A (or a modified form of
Antagonist A) is 5' or 3' end-capped. In other embodiments, the
nucleotide cap is an inverted thymidine.
[0175] VEGF Antagonists
[0176] VEGF antagonists useful in the compositions of the invention
include, but are not limited to, ranibizumab, bevacizumab,
aflibercept, and pharmaceutically acceptable salts thereof.
[0177] In certain embodiments, a VEGF antagonist is an antibody, or
fragment thereof, that binds human VEGF, which may be a humanized
or human anti-VEGF antibody. In particular embodiments, an
anti-VEGF antibody heavy chain variable domain comprises the amino
acid sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYX.sub.1FTX.sub.2YGMNWVRQAPGKGLEWVGWINT
YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPX.sub.3YYG
X.sub.4SHWYFDNvVGQGTLVTVSS (SEQ ID NO:76), wherein X.sub.1 is T or
D; X, is N or H; X.sub.3 is Y or H; and X.sub.4 is S or T. In a
particular embodiment, the heavy chain variable domain comprises
the amino acid sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYT
GEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFD VWGQGTL
(SEQ ID NO:77). These heavy chain variable domain sequences may be
combined with the following light chain variable domain sequences
or with other light chain variable domain sequences, provided that
the antibody so produced binds human VEGF.
[0178] In certain embodiments, an anti-VEGF antibody light chain
variable domain comprises hypervariable regions with the following
amino acid sequences: CDRL1 (SASQDISNYLN [SEQ ID NO:78]), CDRL2
(FTSSLHS [SEQ ID NO:79]) and CDRL3 (QQYSTVPWT [SEQ ID NO:80]). In
particular embodiment, the three light chain hypervariable regions
are provided in a human framework region, e.g., as a contiguous
sequence represented by the following formula:
FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4. In one embodiment, an anti-VEGF
antibody light chain variable domain comprises the amino acid
sequence:
DIQX.sub.1TQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR (SEQ ID
NO:81), wherein X, is M or L. In particular embodiments, the light
chain variable domain comprises the amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID
NO:82). These light chain variable domain sequences may be combined
with the above-identified heavy chain variable domain sequences or
with other heavy chain variable domain sequences, provided that the
antibody so produced retains the ability to bind to human VEGF.
[0179] In one particular embodiment, the VEGF antagonist is the
antibody bevacizumab or a pharmaceutically acceptable salt thereof,
which includes the following heavy and light chain variable domain
sequences, respectively:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYT
GEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFD VWGQGTL
(SEQ ID NO:77); and
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID
NO:82). Bevacizumab is commercially available under the trademark
Avastiniv.RTM. (Genentech, S. San Francisco, Calif.) and is also
described in U.S. Pat. No. 6,054,297.
[0180] In certain embodiments, the VEGF antagonist is a variant of
a parent anti-VEGF antibody (which parent is optionally a humanized
or human anti-VEGF antibody), wherein the variant binds human VEGF
and comprises an amino acid substitution in a hypervariable region
of the heavy or light chain variable domain of the parent anti-VEGF
antibody. In particular embodiments, the variant has one or more
substitution(s) in one or more hypervariable region(s) of the
anti-VEGF antibody. In more particular embodiments, the
substitution(s) are in the heavy chain variable domain of the
parent antibody. For example, the amino acid substitution(s) may be
in the CDRH1 or CDRH3 of the heavy chain variable domain, or there
may be substitutions in both these hypervariable regions. In
certain embodiments, such "affinity matured" variants bind human
VEGF more strongly than the parent anti-VEGF antibody from which
they are generated, i.e., they have a K.sub.d value which is
significantly less than that of the parent anti-VEGF antibody. In
certain embodiments, the variant has an ED50 value for inhibiting
VEGF-induced proliferation of endothelial cells in vitro which is
at least about 10 fold lower, at least about 20 fold lower, or at
least about 50 fold lower, than that of the parent anti-VEGF
antibody. In one embodiment, a variant has a CDRH1 comprising the
amino acid sequence: GYDFTHYGMN (SEQ ID NO:83) and a CDRH3
comprising the amino acid sequence: YPYYYGTSHWYFDV (SEQ ID NO:84).
These hypervariable regions and CDRH2 may be provided in a human
framework region, e.g., resulting in a heavy chain variable domain
comprising the following amino acid sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYT
GEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFD VWGQGTL
(SEQ ID NO:77). Such heavy chain variable domain sequences are
optionally combined with a light chain variable domain comprising
the amino acid domain comprising the following amino acid
sequence:
TABLE-US-00003 (SEQ ID NO: 82)
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLI
YFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPW
TFGQGTKVEIKRTV.
[0181] In one embodiment, the VEGF antagonist is the antibody
fragment ranibizumab or a pharmaceutically acceptable salt thereof,
which includes the following heavy and light chain variable domain
sequences, respectively:
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYT
GEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFD VWGQGTL
(SEQ ID NO:77); and
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID NO:82).
Ranibizumab is commercially available under the trademark
Lucentis.RTM., in which it is formulated for intravitreal
administration (Genentech, S. San Francisco, Calif.) and is also
described in U.S. Pat. No. 7,060,269.
[0182] In another embodiment, the VEGF antagonist is a
VEGF-Trap.TM., such as aflibercept or a pharmaceutically acceptable
salt thereof (see Do et al. (2009) Br J Ophthalmol. 93: 144-9,
which is hereby incorporated by reference in its entirety).
Aflibercept is also known by the name VEGF-Trap-Eye.TM. and is
commercially available under the trademark Eylea.TM. (Regeneron
Pharmaceuticals, Tarrytown, N.Y.). In particular embodiments, a
VEGF-Trap.TM. comprises a dimeric fusion polypeptide comprising two
fusion polypeptides, each fusion polypeptide comprising a VEGF
receptor component consisting of an immunoglobulin-like (Ig) domain
2 of a first VEGF receptor human Flt1 and an Ig domain 3 of a
second VEGF receptor human Flk1 to human Flt4. Aflibercept is a
fusion protein comprising Fc fragments of IgG fused to VEGF
receptor 1 domain 2 and VEGF receptor 2 domain 3, which binds both
VEGF-A and Placental Growth Factor (PIGF). Aflibercept is a dimeric
glycoprotein with a protein molecular weight of 97 kilodaltons
(kDa) and contains glycosylation, constituting an additional 15% of
the total molecular mass, resulting in a total molecular weight of
115 kDa. Illustrative VEGF-Traps, including aflibercept, and
methods of producing the same are described in U.S. Pat. Nos.
7,306,799, 7,531,173, 7,608,261, 7,070,959, 7,374,757, and
7,374.758. In particular embodiments, a VEGF-Trap.TM. is a
polypeptide comprising or consisting of the following amino acid
sequence:
TABLE-US-00004 (SEQ ID NO: 85)
MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEG
RELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK
EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLV
LNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTL
TIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVDLSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK.
Compositions
[0183] The present invention provides compositions, including
pharmaceutical compositions, comprising an anti-PDGF aptamer and a
VEGF antagonist. In some embodiments, the compositions provide
stability to anti-PDGF aptamers or VEGF antagonists, or to both
anti-PDGF aptamers and VEGF antagonists, including those useful in
treating or preventing ophthalmological diseases. In particular
embodiments, the one or more anti-PDGF aptamers present in the
composition is the aptamer Antagonist A or a modified form thereof.
In particular embodiments, the one or more VEGF antagonists present
in the composition is one or more of ranibizumab, bevacizumab, and
aflibercept. In particular embodiments, compositions of the
invention comprise: (i) Antagonist A (or a modified form thereof)
and ranibizumab; (ii) Antagonist A (or a modified form thereof) and
bevacizumab; or (iii) Antagonist A (or a modified form thereof) and
aflibcrcept. In certain embodiments, the compositions comprise a
pharmaceutically acceptable salt of any of the anti-PDGF aptamers
or VEGF antagonists. In particular embodiments, at least about 90%
of the anti-PDGF aptamer or VEGF antagonist is chemically stable
when the composition is stored at a temperature of from about
2.0.degree. C. to about 8.0.degree. C. for at least about twelve
weeks.
[0184] The relative concentrations of the anti-PDGF aptamer and the
VEGF antagonist present in a composition of the invention may be
determined based on the strength and specificity of these
antagonists, and the types and concentration of their binding
targets. In one embodiment, the anti-PDGF aptamer and the VEGF
antagonist are present in substantially equal concentration in the
composition. In another embodiment, the anti-PDGF aptamer or the
VEGF antagonist is present in a substantially higher concentration
than the other, e.g, the ratio of the anti-PDGF aptamer:VEGF
antagonist concentrations in a composition is about 1.5:1, about
2:1, about 2.5:1, about 3:1, about 4:1, or about 5:1, or the ratio
of the VEGF antagonist:anti-PDGF aptamer concentrations in a
composition is about 1.5:1, about 2:1, about 2.5:1, about 3:1,
about 4:1, or about 5:1. In certain embodiments, the ratio of the
anti-PDGF aptamer:VEGF antagonist concentration in a composition is
in the range of about 1:1 to about 5:1, about 1.5:1 to about 5:1,
or about 2.0:1 to about 5:1; in other embodiments, the ratio of the
VEGF antagonist:anti-PDGF aptamer concentration in a composition is
in the range of about 1:1 to about 5:1, about 1.5:1 to about 5:1,
or about 2.0:1 to about 5:1. Unless otherwise indicated, the
concentration of an aptamer is based solely on the molecular weight
of the nucleic acid portion of the aptamer, which can optionally
comprise a short-chain polyethylene glycol. Where the nucleic acid
portion comprises a short chain polyethylene glycol, the molecular
weight of the nucleic acid portion includes the molecular weight of
all short chain polyethylene glycol residues.
[0185] In some embodiments, the anti-PDGF aptamer and the VEGF
antagonist are each present in the composition of the invention at
a concentration from about 0.1 mg/mL to about 200 mg/mL, about 1 to
about 150 mg/mL, about 2 mg/mL to about 100 mg/mL, about 3 mg/mL to
about 80 mg/mL, about 4 mg/mL to about 50 mg/mL, about 4 mg/mL to
about 30 mg/mL, about 5 mg/mL to about 25 mg/mL, or about 5 mg/mL
to about 20 mg/mL. In some embodiments, the anti-PDGF aptamer is
present in the composition at a concentration from about 0.1 mg/mL
to about 200 mg/mL, about 1 to about 150 mg/mL, about 2 mg/mL to
about 100 mg/mL, about 3 mg/mL to about 80 mg/mL, about 4 mg/mL to
about 50 mg/mL, about 4 mg/mL to about 30 mg/mL, about 5 mg/mL to
about 25 mg/mL, or about 5 mg/mL to about 20 mg/mL. In some
embodiments, the VEGF antagonist is present in the composition at a
concentration from about 0.1 mg/mL to about 200 mg/mL, about 1 to
about 150 mg/mL, about 2 mg/mL to about 100 mg/mL, about 3 mg/mL to
about 80 mg/mL, about 4 mg/mL to about 50 mg/mL, about 4 mg/mL to
about 30 mg/mL, about 5 mg/mL to about 25 mg/mL, about 10 mg/mL to
about 25 mg/mL, or about 5 mg/mL to about 20 mg/mL. In some
embodiments, the anti-PDGF aptamer and the VEGF antagonist are each
present at a concentration of at least about 0.1 mg/mL, at least
about 1 mg/mL, at least about 2 mg/mL, at least about 3 mg/mL, at
least about 4 mg/mL, at least about 5 mg/mL, at least about 6
mg/mL, at least about 7 mg/mL, at least about 8 mg/mL, at least
about 9 mg/mL, at least about 10 mg/mL, at least about 15 mg/mL, at
least about 20 mg/mL, at least about 30 mg/mL, at least about 40
mg/mL, at least about 50 mg/mL, at least about 60 mg/mL, at least
about 70 mg/mL, at least about 80 mg/mL, at least about 90 mg/mL,
at least about 100 mg/mL, at least about 120 mg/mL, at least about
150 mg/mL or at least about 200 mg/mL. In some embodiments, at
least one of the anti-PDGF aptamer or VEGF antagonist is present at
a concentration of at least about 0.1 mg/mL, at least about 1
mg/mL, at least about 2 mg/mL, at least about 3 mg/mL, at least
about 4 mg/mL, at least about 5 mg/mL, at least about 6 mg/mL, at
least about 7 mg/mL, at least about 8 mg/mL, at least about 9
mg/mL, at least about 10 mg/mL, at least about 15 mg/mL, at least
about 20 mg/mL, at least about 30 mg/mL, at least about 40 mg/mL,
at least about 50 mg/mL, at least about 60 mg/mL, at least about 70
mg/mL, at least about 80 mg/mL, at least about 90 mg/mL, at least
about 100 mg/mL, at least about 120 mg/mL, at least about 150 mg/mL
or at least about 200 mg/mL.
[0186] Compositions of the invention may also comprise one or more
excipients, buffers (i.e., buffering agents), cryoprotectants,
tonicity agents (i.e., tonicity modifiers), liquids, stabilizers,
surfactants (e.g., nonionic surfactants), lyoprotectants,
antioxidants, amino acids, pH-adjusting agents or preservatives,
such as any of those described herein. Suitable buffering agents
include, but are not limited to, monobasic sodium phosphate,
dibasic sodium phosphate, tris(hydroxymethyl)aminomethane (Tris)
and sodium acetate. In certain embodiments, a buffer is capable of
adjusting the pH of a composition to a desired pH or within a
desired pH range, and/or is capable of achieving or maintaining the
pH of a composition at a desired pH or within a desired pH range.
Suitable nonionic surfactants include, but are not limited to,
polyoxyethylene sorbitan fatty acid esters such as polysorbate 20
and polysorbate 80. Suitable preservatives include, but are not
limited to, benzyl alcohol. Suitable tonicity agents include, but
are not limited to sodium chloride, mannitol, and sorbitol.
Suitable lyoprotectants include, but are not limited to, sucrose,
trehalose, and mannitol. Suitable amino acids include, but are not
limited to glycine and histidine. Suitable pH-adjusting agents (or
agents capable of achieving or maintaining a desired pH or pH
range) include, but are not limited to, hydrochloric acid, acetic
acid, and sodium hydroxide. In one embodiment, the pH-adjusting
agent or agents (or agent(s) capable of achieving or maintaining a
desired pH or pH range) are present in an amount effective to
provide a composition with a pH of about 3 to about 8, about 4.0 to
about 8.0, about 4 to about 7, about 5 to about 6, about 6 to about
7, about 6 to about 8, or about 7 to about 7.5. Suitable excipients
for a composition also include those described in U.S. Pat. No.
7,365,166, the contents of which are herein incorporated by
reference in its entirety.
[0187] In particular embodiments, compositions of the invention
comprise the following: (1) an anti-PDGF aptamer; (2) a VEGF
antagonist; (3) a buffer; (4) a tonicity modifier; and, optionally,
(5) a surfactant. In specific embodiments of such compositions, the
buffer is acetate, phosphate, Tris or histidine, or a mixture
thereof; the tonicity modifier is sodium chloride, mannitol,
sorbitol, or trehalose, or a mixture thereof; and the surfactant is
polysorbate 20. In various embodiments, the anti-PDGF aptamer is
present in the composition of the invention at a concentration of
about 0.1 mg/mL to about 200 mg/mL; the VEGF antagonist is present
at a concentration of about 0.1 mg/mL to about 200 mg/mL; the
buffer is present at a concentration of about 1 mM to about 200 mM;
the tonicity modifier is present at a concentration of about 10 mM
to about 200 mM (sodium chloride), about 1% to about 10% (w/v)
(sorbitol), or about 1% to about 20% (w/v) (trehalose); and the
surfactant, when present, is present at a concentration of about
0.005% to about 0.05% or a concentration of about 0.001% to about
0.05%.
[0188] The compositions of the invention are, in one useful aspect,
administered parenterally (e.g., by intramuscular, intraperitoneal,
intravenous, intraocular, intravitreal, retro-bulbar,
subconjunctival, subtenon or subcutaneous injection or implant) or
systemically. Compositions for parenteral or systemic
administration may include sterile aqueous or non-aqueous
solutions, suspensions, or emulsions. A variety of aqueous carriers
can be used, e.g., water, buffered water, saline, and the like.
Examples of other suitable vehicles include polypropylene glycol,
polyethylene glycol, vegetable oils, gelatin, hydrogels,
hydrogenated naphalenes, and injectable organic esters, such as
ethyl oleate. Such compositions may also contain auxiliary
substances, such as preserving, wetting, buffering, emulsifying, or
dispersing agents. Biocompatible, biodegradable lactide polymer,
lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene
copolymers may be used to control the release of the active
ingredients. In one embodiment, a composition comprising an
anti-PDGF aptamer and a VEGF antagonist is in the form of an
aqueous solution that is suitable for injection. In one embodiment,
a composition comprises an anti-PDGF aptamer, a VEGF antagonist, a
buffering agent, a pH-adjusting agent (or agent capable of
achieving or maintaining a desired pH or pH range), and water for
injection.
[0189] In some examples, the compositions of the invention can also
be administered topically, for example, by patch or by direct
application to a region, such as the epidermis or the eye,
susceptible to or affected by a neovascular disorder, or by
iontophoresis.
[0190] Compositions of the invention may be administered
intraocularly by intravitreal injection into the eye as well as by
subconjunctival and subtenon injections. Other routes of
administration include transcleral, retrobulbar, intraperitoneal,
intramuscular, and intravenous. Alternatively, compositions can be
administered using a drug delivery device or an intraocular
implant. Compositions useful for ophthalmic use include
pharmaceutical compositions comprising an anti-PDGF aptamer and a
VEGF antagonist in admixture with a pharmaceutically acceptable
excipient, including those described herein. These excipients may
be, for example, buffers, inert diluents or fillers (e.g., sucrose
and sorbitol), lubricating agents, glidants, and antiadhesives
(e.g., magnesium stearate, zinc stearate, stearic acid, silicas,
hydrogenated vegetable oils, or talc).
[0191] In particular embodiments, compositions of the invention
confer physical or chemical stability to one or more of the
anti-PDGF aptamers or VEGF antagonists present in the composition.
In these embodiments, the compositions of the invention are
physically or chemically stable compositions. For example,
compositions of the invention may render the anti-PDGF aptamer(s)
or VEGF antagonist(s) present in the composition physically or
chemically stable during storage. Various analytical techniques
useful for evaluating the stability of the anti-PDGF aptamer(s) and
VEGF antagonist(s) are available in the art, including those
described in the accompanying Examples, and those reviewed in
Reubsaet et al. (1998) J. Pharm. Biomed. Anal. 17(6-7): 955-78 and
Wang (1999) Int. J. Pharm. 185(2): 129-88, including visual
inspection, SDS-PAGE, IEF, (high pressure) size exclusion
chromatography (HPSEC), RFFIT, kappa/lambda ELISA. Methods
described in the accompanying Examples include SE-HPLC, AEX-HPLC,
and WCX-HPLC.
[0192] An anti-PDGF aptamer's or a VEGF antagonist's physical
stability in a composition of the invention can be determined by,
but not limited to, measuring the aptamer's or antagonist's state
of physical integrity, determining whether it shows any sign of
aggregation, precipitation or denaturation upon visual examination
of color or clarity, or performing UV light scattering or by size
exclusion chromatography (SEC) or differential scanning calorimetry
(DSC). For example, micro-flow analysis can be used to measure the
presence and size of subvisible particles in a composition, e.g.,
as described in Example 4.
[0193] An anti-PDGF aptamer's or a VEGF antagonist's chemical
stability in a composition of the invention can be determined by,
but not limited to, measuring its state of chemical integrity or
determining whether it shows any sign of decomposition or
modification resulting in formation of a new chemical entity.
Chemical integrity can be assessed by detecting and quantifying
chemically altered forms of the aptamer or antagonist. Chemical
alteration may involve size modification (e.g., clipping) which can
be evaluated using size exclusion chromatography, SDS-PAGE, size
exclusion chromatography with HPLC (to determine the presence of
LMW and HMW species) or matrix-assisted laser desorption
ionization/time-of-flight mass spectrometry (MALDLTOF MS), for
example. Suitable systems for making such measurements are known in
the art, e.g., HPLC systems (Waters, Milford, Mass.) and cation
exchange-HPLC (CEX-HPLC to detect variants and monitor surface
charge). In addition, the methods described in the accompanying
Examples useful for measuring stability of anti-PDGF aptamers or
VEGF antagonists may be used. These include SE-HPLC, WCX-HPLC, and
AEX-HPLC. Other types of chemical alteration include charge
alteration (e.g., occurring as a result of deamidation) which can
be evaluated by ion-exchange chromatography, for example. Oxidation
is another chemical modification that can be detected using methods
disclosed herein or methods known to those skilled in the art.
[0194] In particular embodiments, a composition of the invention is
physically stable if at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, a least
about 95%, or at least about 99% of an anti-PDGF aptamer or a VEGF
antagonist present in the composition shows no sign of aggregation,
precipitation or denaturation upon visual examination of color or
clarity, or as measured by UV light scattering or by size exclusion
chromatography (SEC) or differential scanning calorimetry (DSC). In
particular embodiments, a composition is physically stable if at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%/o, at least about 95%, or at least
about 99% of both the anti-PDGF aptamer(s) and the VEGF
antagonist(s) present in the composition show no sign of
aggregation, precipitation or denaturation upon visual examination
of color or clarity, or as measured by UV light scattering or by
size exclusion chromatography (SEC) or differential scanning
calorimetry (DSC).
[0195] In certain embodiments, physical stability may be determined
by micro-flow imaging, where a greater number of particles or
greater size of particles detected generally correlates with
reduced physical stability. In particular embodiments, a
composition of the invention is physically stable if its particle
count as determined by micro-flow imaging, e.g., as described in
Example 4, e.g., is less than about 500,000, less than about
100,000, or less than about 50,000 total particles/mL, where the
particles have an equivalent circular diameter in the range of 0
.mu.m to about 100 .mu.m or, in another embodiment, in the range of
0 .mu.m to about 25 .mu.m. In another embodiment, a composition of
the invention is considered physically stable if its particle count
as determined by micro-flow imaging, e.g., as described in Example
4, e.g., is less than about 100,000, less than about 50,000, less
than about 20,000, less than about 10,000, less than about 5,000,
less than about 2,500, less than about 1,000, or less than about
500 particles/mL, where the particles have an equivalent circular
diameter in the range of about 1 .mu.m to about 2 .mu.m or, in
another embodiment, in the range of about 1 .mu.m to about 5
.mu.m.
[0196] In particular embodiments, a composition of the invention is
chemically stable when at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, a least
about 95%, or at least about 99% of an anti-PDGF aptamer or a VEGF
antagonist present in the composition shows no decomposition or
modification resulting in formation of a new chemical entity.
[0197] In particular embodiments, an anti-PDGF aptamer or a VEGF
antagonist is chemically stable when at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, a least about 95%, or at least about 99% of an anti-PDGF
aptamer or a VEGF antagonist shows no decomposition or modification
resulting in formation of a new chemical entity. In particular
embodiments, a composition of the invention is chemically stable if
at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, a least about 95%, or at least
about 99% of both the anti-PDGF aptamer(s) and the VEGF
antagonist(s) present in the composition show no decomposition or
modification resulting in formation of a new chemical entity. In
certain embodiments, the decomposition or modification is that
which results in formation of a new chemical entity, for example,
by chemical bond cleavage.
[0198] In particular embodiments, a composition of the invention is
chemically stable when at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, a least
about 95%, or at least about 99% of one or more of the anti-PDGF
aptamers or VEGF antagonists in the composition show no sign of
decomposition or modification resulting in formation of a new
chemical entity, when stored at about room temperature for at least
five days, at least seven days, at least 10 days, at least 14 day,
at least 20 days, at least 30 days, at least two weeks, at least
four weeks, at least eight weeks, at least twelve weeks, at least
sixteen weeks, or at least 24 weeks, at least two months, at least
three months, at least four months, at least six months, or at
least about a year, or alternatively for at least about two years,
or alternatively for at least about three years, or alternatively
for at least about four years, or alternatively for at least about
five years; or alternatively at a temperature from about
2.0.degree. C. to about 8.0.degree. C. for at least five days, at
least seven days, at least 10 days, at least 14 day, at least 20
days, at least 30 days, at least 30 days, at least two weeks, at
least four weeks, at least eight weeks, at least twelve weeks, at
least sixteen weeks, at least 24 weeks, at least two months, at
least three months, at least four months, at least six months, or
at least about a year, or alternatively for at least about two
years, or alternatively for at least about three years, or
alternatively for at least about four years, or alternatively for
at least about five years; or alternatively at a temperature of
about 5.0.degree. C. for at least two weeks, at least four weeks,
at least eight weeks, at least twelve weeks, at least sixteen
weeks, at least 24 weeks, at least about one year, or at least
about two years, or alternatively for at least about three years,
or alternatively for at least about four years, or alternatively
for at least about five years. In particular embodiments, a
composition is physically or chemically stable when the anti-PDGF
aptamer(s) and VEGF antagonist(s) present in the composition are
chemically stable. In some embodiments, the compositions of the
invention are stable, i.e., physically or chemically stable, at
about 40.degree. C. for up to or at least one week, up to or at
least two weeks, or up to or at least one month. In some
embodiments, the compositions are stable at about -20.degree. C.
for up to or at least one year, or alternatively up to or least two
years, three years, four years, or five years. In some embodiments,
the compositions are stable at about -80.degree. C. for up to or at
least one year, of alternatively up to or at least two years, three
years, four years, or five years. In certain embodiments, the
compositions of the invention are physically or chemically stable
if their particle count as determined by micro-flow imaging as
described, e.g., in Example 4, e.g., is less than about 500,000,
less than about 100,000, or less than about 50,000 total
particles/mL, where the particles have an equivalent circular
diameter in the range of 0 .mu.m to about 100 .mu.m or, in another
embodiment, in the range of 0 .mu.m to about 25 .mu.m; or is less
than about 100,000, less than about 50,000, less than about 20,000,
less than about 10,000, less than about 5,000, less than about
2,500, less than about 1,000, or less than about 500 particles/mL,
where the particles have an equivalent circular diameter in the
range of 1 .mu.m to 2 .mu.m or, in another embodiment, in the range
of 1 .mu.m to 5 .mu.m, after storage at about 5.degree. C. or about
30.degree. C. for about four hours.
[0199] In particular embodiments, the compositions of the invention
are considered physically or chemically stable if after storage the
average number of particles detected does not exceed about 50
particles/mL, where the particles have a diameter >about 10
.mu.m and does not exceed 5 particles/mL, where the particles have
a diameter >25 .mu.m, as measured by the Light Obscuration
Particle Count Test described in (788) Particulate Matter in
Injections, Revised Bulletin, Official Oct. 1, 2011, The United
States Pharmacopeial Convention. As described therein, this test is
performed using a suitable apparatus based on a principle of light
blockage that allows for an automatic determination of the size of
particles and the number of particles according to size. The
apparatus is calibrated using dispersions of spherical particles of
known sizes from 10 .mu.m to 25 .mu.m. These standard particles are
dispersed in particle-free water. Care is taken to avoid
aggregation of particles during dispersion. The test is carried out
under conditions that limit exposure to extraneous particulate
matter, for example, in a laminar flow cabinet. Glassware and
filtration equipment used, except for the membrane filters, are
carefully washed with a warm detergent solution and rinsed with
abundant amounts of water to remove all traces of detergent.
Immediately before use, the equipment is rinsed from top to bottom,
outside and then inside, with particle-free water. Care is taken to
not introduce air bubbles into the sample to be measured,
especially when fractions of the preparation are being transferred
to the container in which the measurement is to be carried out. In
order to check that the environment is suitable for the test, that
the glassware is properly cleaned, and that the water to be used is
particle-free, the particulate matter in 5 samples of particle-free
water, each of 5 mL, is determined as immediately follows. If the
number of particles of 10 .mu.m or greater exceeds 25 for the
combined 25 mL, the precautions taken for the test are not
sufficient. The preparatory steps are then repeated until the
environment, glassware, and the water are suitable.
[0200] Once the environment, glassware, and water are suitable for
the test, the test is conducted on the test sample. The contents of
the sample are mixed by slowly inverting the sample's container 20
times successively. If necessary, the container's sealing closure,
if any, is cautiously removed. The outer surfaces of the container
are cleaned using a jet of particle-free water and the container's
sealing closure, if any, is removed, avoiding any contamination of
the contents. Gas bubbles are eliminated by appropriate measures
such as allowing the container to stand for 2 minutes or
sonicating.
[0201] For large-volume samples, 25 mL of greater of volume, single
units are tested. For small-volume samples, less than 25 mL of
volume, the contents of 10 or more units are combined in a cleaned
container to obtain a volume of not less than 25 mL; the test
solution may be prepared by mixing the contents of a suitable
number of vials and diluting the resultant mixture to 25 mL with
particle-free water or with an appropriate particle-free solvent
when particle-free water is not suitable. Small-volume parenterals
having a volume of 25 mL or more may be tested individually.
Powders are reconstituted with particle-free water or with an
appropriate particle-free solvent when particle-free water is not
suitable. The number of test samples should be adequate to provide
a statistically significant assessment. For large-volume samples or
for small-volume samples having a volume of 25 mL or more, fewer
than 10 units may be tested, using an appropriate sampling
plan.
[0202] Four portions, not less than 5 mL each, are removed from
each sample, and the number of particles equal to or greater than
10 .mu.m or 25 .mu.m are counted. The result obtained for the first
portion is disregarded, and the mean number of particles for the
preparation being examined is calculated.
[0203] For samples in containers having a nominal volume of more
than 100 mL, the criteria of Test 1.A described herein should be
considered.
[0204] For samples in containers having a nominal volume of 100 mL
or less, the criteria of Test 1.B described herein should be
considered.
[0205] If the average number of particles exceeds the test limits,
the sample should be tested using the Microscopic Particle Count
Test.
[0206] Test 1.A. The sample complies with the test limits if the
average number of particles present in the sample containers tested
does not exceed 25 per mL, where the particles have a diameter that
is equal to or greater than 10 .mu.m, or if the average number of
particles present in the sample containers tested does not exceed 3
per mL, where the particles have a diameter that is equal to or
greater than 25 .mu.m.
[0207] Test 1.B. The sample complies with the test limits if the
average number of particles present in the sample containers tested
does not exceed 6000 per container, where the particles have a
diameter that is equal to or greater than 10 .mu.m, or if the
average number of particles present in the sample containers does
not exceed 600 per container, where the particles have a diameter
that is equal to or greater than 25 .mu.m.
[0208] In particular embodiments, the compositions are considered
physically or chemically stable if after storage the average number
of particles detected does not exceed 50 particles/mL, where the
particles have a diameter >10 .mu.m; does not exceed 5
particles/mL, where the particles have a diameter >25 .mu.m; and
does not exceed 2 particles/mL, where the particles have a diameter
>50 .mu.m, as measured by the microscopic method particle count
test described in (788) Particulate Matter in Injections, Revised
Bulletin. Official Oct. 1, 2011, The United States Pharmacopeial
Convention.
[0209] The Microscopic Particle Count Test is performed using a
suitable binocular microscope, a filter assembly for retaining
particulate matter, and a membrane filter for examination. The
microscope is adjusted to 100.+-.10 magnifications and is equipped
with an ocular micrometer calibrated with an objective micrometer,
a mechanical stage capable of holding and traversing the entire
filtration area of the membrane filter, and two suitable
illuminators to provide episcopic illumination in addition to
oblique illumination. The ocular micrometer is a circular diameter
graticule and consists of a large circle divided by crosshairs into
quadrants, transparent and black reference circles 10 .mu.m and 25
.mu.m in diameter at 100 magnifications, and a linear scale
graduated in 10 .mu.m increments. It is calibrated using a stage
micrometer that is certified by either a domestic or international
standard institution. A relative error of the linear scale of the
graticule within .+-.2% is acceptable. The large circle is
designated the graticule field of view (GFOV). Two illuminators are
used. One is an episcopic brightfield illuminator internal to the
microscope, the other is an external, focusable auxiliary
illuminator that can be adjusted to give reflected oblique
illumination at an angle of 10.degree. to 20.degree.. The filter
assembly for retaining particulate matter consists of a filter
holder made of glass or other suitable material, and is equipped
with a vacuum source and a suitable membrane filter. The membrane
filter is of suitable size, black or dark gray in color, nongridded
or gridded, and 1.0 .mu.m or finer in nominal pore size.
[0210] The test is carried out under conditions that limit exposure
to extraneous particulate matter, for example, in a laminar flow
cabinet. The glassware and filter assembly used, except for the
membrane filter, are carefully washed with a warm detergent
solution, and rinsed with abundant amounts of water to remove all
traces of detergent. Immediately before use, both sides of the
membrane filter and the equipment are rinsed from top to bottom,
outside and then inside, with particle-free water.
[0211] In order to check that the environment is suitable for the
test, that the glassware and the membrane filter are properly
cleaned, and that the water to be used is particle-free, the
following test is carried out: the particulate matter of a 50-mL
volume of particle-free water is determined according to the method
immediately below. If more than 20 particles of 10 .mu.m or larger
in size or if more than 5 particles of 25 .mu.m or larger in size
are present within the filtration area, the precautions taken for
the test are not sufficient. The preparatory steps are repeated
until the environment, glassware membrane filter, and water are
suitable for the test.
[0212] The contents of the samples are mixed by slowly inverting
the sample's container 20 times successively. If necessary, the
container's sealing closure, if any, is cautiously removed. The
outer surfaces of the container opening are cleaned using a jet of
particle-free water and the sealing closure, if any, is removed,
avoiding any contamination of the contents.
[0213] For large-volume samples, single units are tested. For
small-volume samples less than 25 mL in volume, the contents of 10
or more sample containers are combined in a cleaned container; the
test solution may be prepared by mixing the contents of a suitable
number of vials and diluting to 25 mL with particle-free water or
with an appropriate particle-free solvent when particle-free water
is not suitable. Small-volume samples having a volume of 25 mL or
more may be tested individually. Powders for parenteral use are
constituted with particle-free water or with an appropriate
particle-free solvent when particle-free water is not suitable. The
number of test samples should be adequate to provide a
statistically significant assessment. For large-volume samples or
for small-volume samples having a volume of 25 mL or more, fewer
than 10 units may be tested, using an appropriate sampling
plan.
[0214] The inside of the filter holder fitted with the membrane
filter is wetted with several mL of particle-free water. The total
volume of a solution pool or of a single sample container is
transferred to a filtration funnel, and a vacuum is applied. If
needed, a portion of the sample is added stepwise until the entire
volume is filtered. After the last addition of sample, the inner
walls of the filter holder are rinsed by using a jet of
particle-free water. The vacuum is maintained until the surface of
the membrane filter is free from liquid. The membrane filter is
placed in a Petri dish, and the membrane filter is allowed to
air-dry with the cover slightly ajar. After the membrane filter has
been dried, the Petri dish is placed on the stage of the
microscope, the entire membrane filter is scanned under the
reflected light from the illuminating device, and the number of
particles that are equal to or greater than 10 .mu.m and the number
of particles that are equal to or greater than 25 .mu.m are
counted. Alternatively, partial membrane filter count and
determination of the total filter count by calculation can be
performed. The mean number of particles for the preparation to be
examined is determined.
[0215] The particle sizing process with the use of the circular
diameter graticule is carried out by estimating the equivalent
diameter of the particle in comparison with the 10 .mu.m and 25
.mu.m reference circles on the graticule. Thereby the particles are
not moved from their initial locations within the graticule field
of view and are not superimposed on the reference circles for
comparison. The inner diameter of the transparent graticule
reference circles is used to size white and transparent particles,
while dark particles are sized by using the outer diameter of the
black opaque graticule reference circles.
[0216] Amorphous, semiliquid, or otherwise morphologically
indistinct materials that have the appearance of a stain or
discoloration on the membrane filter should not be sized or counted
because these materials show little or no surface relief and
present a gelatinous or film-like appearance. In such cases, the
interpretation of enumeration may be aided by testing a sample of
the solution by the Light Obscuration Particle Count Test.
[0217] For samples in containers having a nominal volume of more
than 100 mL, apply the criteria of Test 2.A.
[0218] For samples in containers having a nominal volume of 100 mL
or less, apply the criteria of Test 2.B.
[0219] Test 2.A. The sample complies with the test limits if the
average number of particles present in the sample containers tested
does not exceed 12 per mL and the particles have a diameter that is
equal to or greater than 10 .mu.m, or the average number of
particles present in the sample containers tested does not exceed 2
per mL and the particles have a diameter that is equal to or
greater than 25 .mu.m.
[0220] Test 2.B. The sample complies with the test limits if the
average number of particles present in the sample containers tested
does not exceed 3000 per container and the particles have a
diameter that is equal to or greater than 10 .mu.m, or the average
number of particles present in the sample containers tested does
not exceed 300 per container and the particles have a diameter that
is equal to or greater than 25 .mu.m.
[0221] In certain embodiments, the compositions of the invention
are in lyophilized form.
[0222] Compositions Comprising Antagonist A and Ranibizumab
[0223] In certain embodiments, a composition of the invention
comprises Antagonist A or a modified form thereof and ranibizumab.
In particular embodiments, the ratio of the concentration (mass of
Antagonist A less that of its --R group/volume of composition) of
Antagonist A or modified form thereof to the concentration
(mass/volume of composition) of ranibizumab present in the
composition is less than 25.0, less than 10.0, less than 9.0, less
than 8.0, less than 7.0, less than 6.0, less than 5.0, less than
4.0, less than 3.0, less than 2.0 or less than 1.0. In particular
embodiments, the ratio of the concentration (mass of Antagonist A
less that of its --R group/volume of composition) of Antagonist A
or modified form thereof to the concentration (mass/volume of
composition) of ranibizumab present in the composition is less than
or equal to 25.0, less than or equal to 10.0, less than or equal to
9.0, less than or equal to 8.0, less than or equal to 7.0, less
than or equal to 6.0, less than or equal to 5.0, less than or equal
to 4.0, less than or equal to 3.0, less than or equal to 2.0 or
less than or equal to 1.0. In particular embodiments, the ratio of
the concentration (mass of Antagonist A less that of its --R
group/volume of composition) of Antagonist A or modified form
thereof to the concentration (mass/volume of composition) of
ranibizumab present in the composition is in the range of about 1
to about 10, about 2 to about 5, about 3 about 4, or about 5.
[0224] Antagonist A's--R group is depicted in FIG. 78A.
[0225] In particular embodiments, a composition of the invention
comprises Antagonist A or a modified form thereof and ranibizumab,
and the composition is stable with respect to both active agents at
a particular pH or suitable for parenteral administration. In
certain embodiments, the composition comprises one or more of a
tonicity modifier, a surfactant, and a buffer suitable to achieve
or maintain the particular pH or be suitable for parenteral
administration. Appropriate buffers include those described herein
as well as others known in the art, such as, e.g., a Good's
buffers, e.g., MES.
[0226] In certain embodiments, the concentration of Antagonist A or
modified form thereof in the composition of the invention is less
than or about 100 mg/mL, less than about 50 mg/mL, less than about
40 mg/mL, less than about 30 mg/mL, less than about 25 mg/mL, less
than about 20 mg/mL, less than about 15 mg/mL, less than about 10
mg/mL, or less than about 5 mg/mL. In certain embodiments, the
concentration of Antagonist A or modified form thereof is about 0.3
mg/mL to about 100 mg/mL, about 0.3 mg/mL to about 50 mg/mL, about
0.3 mg/mL to about 40 mg/mL, about 0.3 mg/mL to about 30 mg/mL,
about 0.3 to about 25 mg/mL, about 0.3 mg/mL to about 20 mg/mL,
about 0.3 mg/mL to about 15 mg/mL, about 0.3 mg/mL to about 10
mg/mL, about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 50
mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 30
mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 20
mg/mL, about 1 mg/mL to about 15 mg/mL, about 1 mg/mL to about 10
mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 100
mg/mL, or about 5 mg/mL to about 50 mg/mL. In certain embodiments,
the concentration of Antagonist A or modified form thereof is about
1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5
mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL,
about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 24 mg/mL,
about 25 mg/mL, about 30 mg/mL, about 40 mg/mL, or about 50
mg/mL.
[0227] In certain embodiments, the concentration of ranibizumab in
the composition of the invention is about 0.5 mg/mL to about 50
mg/mL, about 0.5 mg/mL to about 20 mg/mL, about 1.0 mg/mL to about
50 mg/mL, about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about
10 mg/mL, or about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11
mg/mL, or about 12 mg/mL.
[0228] In certain embodiments, the concentration of ranibizumab in
the composition of the invention is about 0.5 mg/mL to about 50
mg/mL, about 0.5 mg/mL to about 20 mg/mL, about 1.0 mg/mL to about
50 mg/mL, about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about
10 mg/mL, or about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11
mg/mL, or about 12 mg/mL, and the concentration of Antagonist A or
modified form thereof in the composition is less than about 100
mg/mL, less than about 50 mg/mL, less than about 40 mg/mL, less
than about 30 mg/mL, less than about 25 mg/mL, less than about 20
mg/mL, less than about 15 mg/mL, less than about 10 mg/mL, or less
than about 5 mg/mL.
[0229] In certain embodiments, the concentration of ranibizumab in
the composition of the invention is about 0.5 mg/mL to about 50
mg/mL, about 0.5 mg/mL to about 20 mg/mL, about 1 mg/mL to about 50
mg/mL, about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about 10
mg/mL, or about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11
mg/mL, or about 12 mg/mL, and the concentration of Antagonist A or
modified form thereof is about 0.3 mg/mL to about 100 mg/mL, 0.3
mg/mL to about 50 mg/mL, about 0.3 mg/mL to about 40 mg/mL, about
0.3 mg/mL to about 30 mg/mL, about 0.3 to about 25 mg/mL, about 0.3
mg/mL to about 20 mg/mL, about 0.3 mg/mL to about 15 mg/mL, about
0.3 mg/mL to about 10 mg/mL, about 1.0 mg/mL to about 100 mg/mL,
about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL,
about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 25 mg/mL,
about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 15 mg/mL,
about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 5 mg/mL,
about 5 mg/mL to about 100 mg/mL, or about 5 mg/mL to about 50
mg/mL.
[0230] In certain embodiments, the concentration of ranibizumab in
the compositions of the invention is about 0.5 mg/mL to about 50
mg/mL, about 0.5 mg/mL to about 20 mg/mL, about 1 mg/mL to about 50
mg/mL, about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about 50
mg/mL, about 2 mg/mL to about 10 mg/mL, or about 4 mg/mL, about 5
mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL,
or about 10 mg/mL, and the concentration of Antagonist A or
modified form thereof is about 1 mg/mL, about 2 mg/mL, about 3
mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL,
about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about
20 mg/mL, about 24 mg/mL, about 25 mg/mL, about 30 mg/mL, about 40
mg/mL, or about 50 mg/mL. In one embodiment, the concentration of
Antagonist A or modified form thereof is about 3 mg/mL, and the
concentration of ranibizumab is about 5 mg/mL. In one embodiment,
the concentration of Antagonist A or modified form thereof is about
6 mg/mL, and the concentration of ranibizumab is about 10 mg/mL. In
one embodiment, the concentration of Antagonist A or modified form
thereof is about 15 mg/mL, and the concentration of ranizumab is
about 5 mg/mL. In one embodiment, the concentration of Antagonist A
or modified form thereof is about 24 mg/mL, and the concentration
of ranizumab is about 8 mg/mL.
[0231] In certain embodiments of a composition comprising
Antagonist A or modified form thereof and ranibizumab, the
composition further comprises a tonicity modifier that is sorbitol
or sodium chloride, or mixtures thereof. In particular embodiments,
the tonicity modifier is sorbitol, and the pH of the composition is
about 5.0 to about 8.0, about 5.0 to about 7.0, about 6.0 or about
7.0. In particular embodiments, the tonicity modifier is sodium
chloride, and the pH of the composition is about 5.0 to about 8.0,
about 5.0 to about 7.0, about 5.5 to about 7.5, about 6.0 to about
8.0, about 8.0, about 7.0, or about 6.0. In certain embodiments,
the tonicity modifier is sorbitol at about 1% to about 10% (w/v),
or about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v),
about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v),
about 9% (w/v), or about 10% (w/v). In particular embodiments, the
tonicity modifier is sodium chloride at a concentration of about 10
mM to about 200 mM, about 50 mM to 200 mM, about 75 mM to about 200
mM, about 50 mM to about 150 mM, about 100 mM, about 110 mM, about
120 mM, about 130 mM about 140 mM or about 150 mM. In one
embodiment, the tonicity modifier is sodium chloride at a
concentration of about 130 mM. In other embodiments, the tonicity
modifier is sodium chloride at a concentration of about 75 mM or
about 120 mM. With respect to tonicity modifier concentration, "mM"
refers to milimoles of the tonicity modifier per liter of
composition.
[0232] In certain embodiments of a composition of the invention
comprising Antagonist A or a modified form thereof and ranibizumab,
the composition further comprises a buffer capable of achieving or
maintaining the pH of the composition within a desired range. In
certain embodiments, the composition comprises histidine (e.g.,
L-histidine or a pharmaceutically acceptable salt thereof) or
phosphate as a buffer, e.g., sodium phosphate of potassium
phosphate (or both histidine and phosphate). In certain
embodiments, the buffer is present at a concentration of about 1 mM
to about 200 mM, about 1 mM to about 150 mM, about 1 mM to about 20
mM, about 1 mM to about 10 mM, about 2 mM to about 100 mM, about 2
mM to about 20 mM, about 5 mM to about 20 mM, or about 10 mM. In
particular embodiments, the pH of the buffered composition is about
5.0 to about 8.0, about 5.0 to about 7.0, about 5.5 to about 7.5,
about 5.5 to about 7.0, or about 6.0. In one embodiment, the
buffered composition has a pH of about 5.5 to about 7.0. In certain
embodiments, the buffer comprises histidine at a concentration of
about 1 mM to about 200 mM, about 1 mM to about 150 mM, about 2 mM
to about 100 mM, about 5 mM to about 20 mM, or about 10 mM, and the
buffered composition has a pH of about 5.5 to about 7.0, or about
6.0. In one particular embodiment, the buffer comprises histidine
at a concentration of about 10 mM and the pH of the
histidine-buffered composition is about 6.0. With respect to buffer
concentration, "mM" refers to millimoles of buffer (e.g.,
histidine) per liter of composition.
[0233] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and ranibizumab, the buffer
comprises phosphate, alone or in combination with histidine. The
phosphate buffer may be, e.g., a sodium phosphate or a potassium
phosphate buffer. In certain embodiments, the buffer comprises
phosphate at a concentration of about 1 mM to about 200 mM, about 1
mM to about 50 mM, about 2 mM to about 200 mM, about 2 mM to about
50 mM, about 5 mM to about 200 mM, about 5 mM to about 100 mM,
about 5 mM to about 50 mM, about 10 mM to about 150 mM, about 10 mM
to about 100 mM, about 5 mM, about 10 mM, about 25 mM, or about 50
mM. In particular embodiments, the pH of the buffered composition
is about 5.0 to about 8.0, about 6.0 to about 8.0, about 5.5 to
about 7.5, about 5.5 to about 7.0, about 6.0, about 7.0, or about
8.0. In one embodiment, the buffer comprises phosphate, and the
buffered composition has a pH of about 6.0 to about 8.0. In certain
embodiments, the buffer comprises phosphate at a concentration of
about 5 mM to about 200 mM, about 5 mM to about 150 mM, about 5 mM
to about 100 mM, about 5 mM, about 8 mM, about 10 mM, about 25 mM,
or about 50 mM, and the buffered composition has a pH of about 5.5
to about 7.5, about 5.5 to about 7.0, or about 6.0. In one
particular embodiment, the buffer comprises phosphate at a
concentration of about 10 mM, and the buffered composition has a pH
of about 6.2.
[0234] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and ranibizumab, the
composition further comprises a surfactant. In particular
embodiments, the surfactant is polysorbate 20 at a concentration of
about 0.001% (w/v) to about 0.05% (w/v), about 0.002% (w/v) to
about 0.05% (w/v), about 0.005% (w/v) to about 0.05% (w/v), about
0.01% (w/v) to about 0.05% (w/v), or about 0.02% (w/v).
[0235] In one embodiment, a composition comprises Antagonist A or a
modified form thereof, ranibizumab, histidine, and NaCl. The
composition may further comprise polysorbate.
[0236] In one particular embodiment, a composition of the invention
comprises Antagonist A or a modified version thereof and
ranibizumab; the ratio of the concentration of Antagonist A (or
modified form thereof) to the concentration of ranibizumab is less
than 2; and the composition further comprises sodium chloride at a
concentration of about 10 mM to about 200 mM, histidine at a
concentration of about 1 mM to about 100 mM, and polysorbate (e.g.,
polysorbate 20) at a concentration of about 0.005% to about 0.05%,
where the pH of the composition is about 5.5 to about 7.0.
[0237] In certain embodiments, the present invention provides
compositions comprising Antagonist A or a modified form thereof, or
a pharmaceutically acceptable salt thereof, and ranibizumab, or a
pharmaceutically acceptable salt thereof. In certain embodiments, a
composition of the invention comprises: (a) about 0.3 mg/mL to
about 30 mg/mL Antagonist A or modified form thereof, or a
pharmaceutically acceptable salt thereof; and (b) about 0.5 mg/mL
to about 20 mg/mL ranibizumab or pharmaceutically acceptable salt
thereof. In other embodiments, the compositions further comprise
one or both of: (c) about 1 mM to about 20 mM L-histidine; and (d)
about 10 mM to about 200 mM NaCl. In further embodiments, the
compositions further comprise: (e) about 0.001% (w/v) to about
0.05% (w/v) surfactant, which is optionally polysorbate. In a
particular embodiment, the compositions comprise: (a) about 0.3
mg/mL to about 30 mg/mL Antagonist A or modified form thereof, or
pharmaceutically acceptable salt thereof; (b) about 0.5 mg/mL to
about 20 mg/mL ranibizumab or pharmaceutically acceptable salt
thereof; (c) about 1 mM to about 20 mM L-histidine; and (d) about
10 mM to about 200 mM NaCl, wherein the pH of the compositions is
about pH 5.0 to about pH 7.0. In a further embodiment, the
compositions comprise: (a) about 3 mg/mL Antagonist A or modified
form thereof, or pharmaceutically acceptable salt thereof; (b)
about 5 mg/mL ranibizumab or pharmaceutically acceptable salt
thereof; (c) about 10 mM L-histidine; and (d) about 130 mM NaCl,
wherein the pH of the compositions is about pH 6.0. In certain
embodiments, the compositions further comprise: (e) about 0.01%
(w/v) polysorbate 20.
[0238] In certain embodiments, compositions of the invention
comprise: (a) about 1.0 mg/mL to about 100 mg/mL, or about 5.0
mg/mL to about 50 mg/mL. Antagonist A or modified form thereof, or
a pharmaceutically acceptable salt thereof; and (b) about 1.0 mg/mL
to about 50 mg/mL ranibizumab or pharmaceutically acceptable salt
thereof. In other embodiments, the compositions further comprise
one or both of (c) about 1 mM to about 20 mM L-histidine; and (d)
about 10 mM to about 200 mM NaCl. In further embodiments, the
compositions further comprise: (e) about 0.001% (w/v) to about
0.05% (w/v) surfactant, which is optionally polysorbate. In a
particular embodiment, the compositions comprise: (a) about 5.0
mg/mL to about 50 mg/mL Antagonist A or modified form thereof, or
pharmaceutically acceptable salt thereof; (b) about 1.0 mg/mL to
about 50 mg/mL ranibizumab or pharmaceutically acceptable salt
thereof; (c) about 1 mM to about 20 mM L-histidine; and (d) about
10 mM to about 200 mM NaCl, wherein the pH of the compositions is
about pH 5.0 to about pH 8.0 or about pH 5.5 to about pH 7.5. In a
further embodiment, the compositions comprise: (a) about 15 mg/mL
Antagonist A or modified form thereof, or pharmaceutically
acceptable salt thereof; (b) about 5 mg/mL ranibizumab or
pharmaceutically acceptable salt thereof; (c) about 5 mM
L-histidine; and (d) about 75 mM NaCl, wherein the pH of the
compositions is about pH 5.5 to about pH 7.5 or about pH 6.0. In
certain embodiments, the compositions further comprise: (e) about
0.005% (w/v) polysorbate 20. In a further embodiment, the
compositions comprise: (a) about 24 mg/mL Antagonist A or modified
form thereof, or pharmaceutically acceptable salt thereof; (b)
about 8 mg/mL ranibizumab or pharmaceutically acceptable salt
thereof; (c) about 2 mM L-histidine; and (d) about 120 mM NaCl,
wherein the pH of the compositions is about pH 5.5 to about pH 7.5
or about pH 6.0. In certain embodiments, the compositions further
comprise: (e) about 0.002% (w/v) polysorbate 20.
[0239] In certain embodiments, compositions of the invention
comprise: (a) about 0.3 mg/mL to about 30 mg/mL Antagonist A or a
modified form thereof, or a pharmaceutically acceptable salt
thereof; (b) about 0.5 mg/mL to about 20 mg/mL ranibizumab; and one
or both of (c) a buffer capable of achieving or maintaining the pH
of the composition to about pH 5.0 to about pH 8.0; and (d) a
tonicity modifier. In particular embodiments, the buffer, where
present, is about 1 mM to about 20 mM L-histidine or about 1 mM to
about 20 mM sodium phosphate; and the tonicity modifier, where
present, is about 10 mM to about 200 mM NaCl, about 1% to about 20%
(w/v) sorbitol, or about 1% to about 20% (w/v) trehalose. In
certain embodiments, the buffer is about 1 mM to about 20 mM
L-histidine; and the tonicity modifier is about 10 mM to about 200
mM NaCl, wherein the pH of the composition is about pH 5.0 to about
pH 7.0.
[0240] Any of the compositions of the invention may also comprise a
surfactant, e.g., about 0.001% (w/v) to about 0.05% (w/v)
surfactant.
[0241] Examples of compositions of the invention include the
compositions described in Table 1. Table 3 or Table 8. In other
embodiments, the invention includes the compositions described in
Table 1 but without the polysorbate.
[0242] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof at a concentration of about
3 mg/mL, ranibizumab at a concentration of about 5 mg/mL, histidine
at a concentration of about 10 mM, sodium chloride at a
concentration of about 130 mM and polysorbate 20 at a concentration
of about 0.02% (w/v), wherein the pH of the composition is about
6.0.
[0243] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A or modified form thereof, about 5 mg/mL
ranibizumab, about 10 mM sodium phosphate, about 5% (w/v) sorbitol,
and about 0.01% (w/v) polysorbate 20, wherein the pH of the
composition is about pH 7.0.
[0244] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A or modified form thereof, about 5 mg/mL
ranibizumab, about 10 mM sodium phosphate, about 130 mM NaCl, and
about 0.01% (w/v) polysorbate 20, wherein the pH of the composition
is about pH 7.0.
[0245] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A or modified form thereof, about 5 mg/mL
ranibizumab, about 5 mM sodium phosphate, about 5 mM histidine HCl,
about 75 mM NaCl, about 5% (w/v) trehalose, and about 0.005% (w/v)
polysorbate 20, wherein the pH of the composition is about pH
6.5.
[0246] In certain embodiments the compositions of the invention
comprise: (a) about 3 mg/mL to about 90 mg/mL Antagonist A or a
modified form thereof; (b) about 1.0 mg/mL to about 30 mg/mL
ranibizumab; and one or both of (c) a buffer capable of achieving
or maintaining the pH of the composition to about pH 5.0 to about
pH 8.0; and (d) a tonicity modifier. In particular embodiments, the
buffer, where present, comprises about 1 mM to about 100 mM sodium
phosphate or about 1.0 mM to about 10 mM histidine.HCl; and the
tonicity modifier, where present, is about 0.5% (w/v) to about 10%
(w/v) trehalose.
[0247] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof at a concentration of about
15 mg/mL, ranibizumab at a concentration of about 5 mg/mL,
histidine at a concentration of about 5 mM, sodium chloride at a
concentration of about 75 mM and polysorbate 20 at a concentration
of about 0.005% (w/v), wherein the pH of the composition is about
5.5 to about 7.5.
[0248] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof at a concentration of about
24 mg/mL, ranibizumab at a concentration of about 8 mg/mL,
histidine at a concentration of about 2 mM, sodium chloride at a
concentration of about 120 mM and polysorbate 20 at a concentration
of about 0.002% (w/v), wherein the pH of the composition is about
5.5 to about 7.5.
[0249] In particular embodiments, a composition comprising
Antagonist A or a modified form thereof and ranibizumab is
chemically stable for at least eight weeks or at least twelve weeks
at 25.degree. C. or for at least twelve weeks or at least sixteen
weeks or at least 24 weeks at 4.degree. C. In particular
embodiments, at least 80% of each of Antagonist A and ranibizumab
show no sign of decomposition or modification resulting in
formation of a new chemical entity under at least one of these
conditions.
[0250] Compositions Comprising Antagonist A and Bevacizumab
[0251] In certain embodiments, a composition of the invention
comprises Antagonist A or a modified form thereof and bevacizumab.
In particular embodiments, the ratio of the concentration (mass of
Antagonist A less that of its --R group/volume of composition) of
Antagonist A (or modified form thereof) to the concentration
(mass/volume of composition) of bevacizumab present in the
composition is less than 25.0, less than 10.0, less than 9.0, less
than 8.0, less than 7.0, less than 6.0, less than 5.0, less than
4.0, less than 3.0, less than 2.0, less than 1.0, or less than
0.5.
[0252] Antagonist A's --R group is depicted in FIG. 78A.
[0253] In particular embodiments, a composition of the invention
comprises Antagonist A or a modified form thereof and bevacizumab,
and the composition is stable with respect to both active agents at
a particular pH suitable for parenteral administration. In certain
embodiments, the composition comprises one or more tonicity
modifier, surfactant, and buffer suitable to achieve or maintain
the particular pH or be suitable for parenteral administration.
Appropriate buffers include those described herein as well as
others known in the art, such as, e.g., a Good's buffers, e.g.,
MES.
[0254] In certain embodiments, the concentration of Antagonist A or
modified form thereof in the composition is less than about 50
mg/mL, less than about 40 mg/mL, less than about 30 mg/mL, less
than about 25 mg/mL, less than about 20 mg/mL, less than about 15
mg/mL, less than about 10 mg/mL, or less than about 5 mg/mL. In
certain embodiments, the concentration of Antagonist A or modified
form thereof is about 0.3 mg/mL to about 50 mg/mL, about 0.3 mg/mL
to about 40 mg/mL, about 0.3 mg/mL to about 30 mg/mL, about 0.3 to
about 25 mg/mL, about 0.3 mg/mL to about 20 mg/mL, about 0.3 mg/mL
to about 15 mg/mL, about 0.3 mg/mL to about 10 mg/mL, about 1 mg/mL
to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL
to about 30 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL
to about 20 mg/mL, about 1 mg/mL to about 15 mg/mL, about 1 mg/mL
to about 10 mg/mL, or about 1 mg/mL to about 5 mg/mL. In certain
embodiments, the concentration of Antagonist A or modified form
thereof is about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4
mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL,
about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL,
about 25 mg/mL, about 30 mg/mL, about 40 mg/mL, or about 50
mg/mL.
[0255] In certain embodiments, the concentration of bevacizumab is
about 0.5 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about 25
mg/mL, about 1 mg/mL to about 50 mg/mL, about 1.0 to about 25
mg/mL, about 1.0 to about 20 mg/mL, about 5 mg/mL to about 50
mg/mL, about 5 mg/mL to about 25 mg/mL, about 5 mg/mL to about 25
mg/mL, about 5 mg/mL to about 20 mg/mL, about 12.5 mg/mL, about 25
mg/mL, or about 50 mg/mL.
[0256] In certain embodiments, the concentration of bevacizumab is
about 0.5 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about 25
mg/mL, about 1 mg/mL to about 50 mg/mL, about 1.0 to about 25
mg/mL, about 1.0 to about 20 mg/mL, about 5 mg/mL to about 50
mg/mL, about 5 mg/mL to about 25 mg/mL, about 5 mg/mL to about 25
mg/mL, about 5 mg/mL to about 20 mg/mL, about 12.5 mg/mL, about 25
mg/mL, or about 50 mg/mL, and the concentration of Antagonist A or
modified form thereof is less than about 50 mg/mL, less than about
40 mg/mL, less than about 30 mg/mL, less than about 25 mg/mL, less
than about 20 mg/mL, less than about 15 mg/mL, less than about 10
mg/mL, or less than about 5 mg/mL.
[0257] In certain embodiments, the concentration of bevacizumab is
about 0.5 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about 25
mg/mL, about 1 mg/mL to about 50 mg/mL, about 1.0 to about 25
mg/mL, about 1.0 to about 20 mg/mL, about 5 mg/mL to about 50
mg/mL, about 5 mg/mL to about 25 mg/mL, about 5 mg/mL to about 25
mg/mL, about 5 mg/mL to about 20 mg/mL, about 12.5 mg/mL, about 25
mg/mL, or about 50 mg/mL, and the concentration of Antagonist A or
modified form thereof is about 0.3 mg/mL to about 50 mg/mL, about
0.3 mg/mL to about 40 mg/mL, about 0.3 mg/mL to about 30 mg/mL,
about 0.3 to about 25 mg/mL, about 0.3 mg/mL to about 20 mg/mL,
about 0.3 mg/mL to about 15 mg/mL, about 0.3 mg/mL to about 10
mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40
mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 25
mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 15
mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 5
mg/mL.
[0258] In certain embodiments, the concentration of bevacizumab is
about 0.5 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about 25
mg/mL, about 1 mg/mL to about 50 mg/mL, about 1.0 to about 25
mg/mL, about 1.0 to about 20 mg/mL, about 5 mg/mL to about 50
mg/mL, about 5 mg/mL to about 25 mg/mL, about 5 mg/mL to about 25
mg/mL, about 5 mg/mL to about 20 mg/mL, about 12.5 mg/mL, about 25
mg/mL, or about 50 mg/mL, and the concentration of Antagonist A or
modified form thereof is about 1 mg/mL, about 2 mg/mL, about 3
mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL,
about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about
20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 40 mg/mL, or about
50 mg/mL. In one embodiment, the concentration of Antagonist A or
modified form thereof is about 3 mg/mL and the concentration of
bevacizumab is about 12.5 mg/mL. In another embodiment, the
concentration of Antagonist A or modified form thereof is about 6
mg/mL, and the concentration of bevacizumab is about 25 mg/mL or
about 50 mg/mL.
[0259] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and bevacizumab, the
composition further comprises a tonicity modifier selected from
sorbitol, sodium chloride and trehalose. In other embodiments, the
composition comprises both sorbitol and sodium chloride, both
sodium chloride and trehalose, or both sorbitol and trehalose. In
particular embodiments, the composition comprises sorbitol, and the
pH of the composition is about 7.0 to about 8.0. In particular
embodiments, the composition comprises sodium chloride, and the pH
of the composition is about 6.0 to about 8.0. In certain
embodiments, the composition comprises trehalose, and the pH of the
composition is about 6.0 to about 7.0. In certain embodiments, the
composition comprises sorbitol at about 1% to about 10% (w/v), or
about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v),
about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v),
about 9% (w/v), or about 10% (w/v). In particular embodiments, the
composition comprises sodium chloride at a concentration of about
10 mM to about 200 mM, about 50 mM to 200 mM, about 75 mM to about
200 mM, about 50 mM to about 150 mM, about 100 mM, about 110 mM,
about 120 mM, about 130 mM about 140 mM or about 150 mM. In one
embodiment, the composition comprises sodium chloride at a
concentration of about 130 mM. In certain embodiments, the
composition comprises trehalose at about 1% to about 10% (w/v), or
about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v),
about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v),
about 9% (w/v), or about 10% (w/v).
[0260] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and bevacizumab, the
composition further comprises a buffer capable of achieving or
maintaining the pH of the composition within a desired range. In
certain embodiments, the composition comprises one or more of
acetate, phosphate, and Tris as a buffer. In certain embodiments,
the buffer comprises phosphate at a concentration of about 5 mM to
about 200 mM, about 5 mM to about 100 mM, about 10 mM to about 150
mM, about 10 mM to about 100 mM, about 25 mM to about 100 mM, or
about 50 mM. The phosphate buffer may be, e.g., a sodium phosphate
buffer or a potassium phosphate buffer. In particular embodiments,
the pH of the buffered composition is about 5.0 to about 8.0, about
6.0 to about 8.0, about 5.5 to about 7.0, about 6.0, about 7.0, or
about 8.0. In one embodiment, the buffer comprises phosphate, and
the pH of the buffered composition is about 5.5 to about 7.0. In
certain embodiments, the buffer comprises phosphate at a
concentration of about 5 mM to about 200 mM, about 10 mM to about
150 mM, about 25 mM to about 100 mM, or about 50 mM, and the
buffered composition has a pH of about 5.5 to about 7.0, or about
6.0. In one particular embodiment, the buffer comprises phosphate
at a concentration of about 50 mM, and the buffered composition has
a pH of about 6.0.
[0261] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and bevacizumab, the
composition further comprises a surfactant. In particular
embodiments, the surfactant is polysorbate 20 at a concentration of
about 0.005% (w/v) to about 0.05% (w/v), about 0.01% (w/v) to about
0.05% (w/v), or about 0.02% (w/v).
[0262] In one embodiment, a composition comprising Antagonist A or
a modified form thereof and bevacizumab comprises Antagonist A,
bevacizumab, sodium chloride, and phosphate. The composition may
further comprise polysorbate.
[0263] In one particular embodiment, a composition comprises
Antagonist A or a modified form thereof and bevacizumab; the ratio
of the concentration of Antagonist A (or modified form thereof) to
the concentration of bevacizumab is less than 1.5, less than 1.2 or
less than 1; and the composition further comprises sodium chloride
at a concentration of about 10 mM to about 200 mM, phosphate at a
concentration of about 5 mM to about 200 mM, and polysorbate (e.g.,
polysorbate 20) at a concentration of about 0.005% to about 0.05%,
wherein the pH of the composition is about 5.5 to about 7.0.
[0264] In certain embodiments, the present invention provides
compositions comprising Antagonist A or a modified form thereof, or
a pharmaceutically acceptable salt thereof, and bevacizumab, or a
pharmaceutically acceptable salt thereof. In certain embodiments, a
composition of the invention comprises: (a) about 0.3 mg/mL to
about 30 mg/mL Antagonist A or a modified form thereof, or
pharmaceutically acceptable salt thereof; and (b) about 0.5 mg/mL
to about 25 mg/mL bevacizumab or pharmaceutically acceptable salt
thereof. In other embodiments, the composition further comprises
one or both of (c) about 5 mM to about 200 mM phosphate buffer; and
(d) about 10 mM NaCl to about 200 mM NaCl. In other embodiments,
the composition comprises: (a) about 0.3 mg/mL to about 30 mg/mL
Antagonist A or modified form thereof, or pharmaceutically
acceptable salt thereof; (b) about 0.5 mg/mL to about 25 mg/mL
bevacizumab or pharmaceutically acceptable salt thereof; (c) about
5 mM to about 200 mM phosphate buffer, (e.g., about 5 mM to about
200 mM sodium phosphate); and (d) about 10 mM NaCl to about 200 mM
NaCl, wherein the pH of the composition is about pH 5.0 to about pH
7.0. In particular embodiments of compositions comprising
bevacizumab, the composition further comprises: (e) about 0.001%
(w/v) to about 0.05% (w/v) surfactant, which is optionally
polysorbate. In a particular embodiment, the composition comprises:
(a) about 3 mg/mL Antagonist A or modified form thereof, or
pharmaceutically acceptable salt thereof; (b) about 12.5 mg/mL
bevacizumab or pharmaceutically acceptable salt thereof; (c) about
50 mM phosphate buffer; and (d) about 130 mM NaCl, wherein the pH
of the composition is about pH 6.0. In another embodiment, the
composition further comprises: (e) about 0.01% (w/v) polysorbate
20.
[0265] In certain embodiments, the compositions of the invention
comprise: (a) about 0.3 mg/mL to about 30 mg/mL Antagonist A or a
modified form thereof; (b) about 0.5 mg/mL to about 25 mg/mL
bevacizumab; and one or both of (c) a buffer capable of achieving
or maintaining the pH of the composition to about pH 5.0 to about
pH 8.0; and (d) a tonicity modifier. In particular embodiments, the
buffer is about 5 mM to about 200 mM sodium phosphate or about 5 mM
to about 200 mM Tris.HCl; and the tonicity modifier is about 10 mM
to about 200 mM NaCl, about 1% to about 20% (w/v) sorbitol, or
about 1% to about 20% (w/v) trehalose. In certain embodiments, the
buffer is about 5 mM to about 200 mM sodium phosphate; and the
tonicity agent is about 10 mM to about 200 mM NaCl, wherein the pH
of the composition is about pH 5.0 to about pH 7.0. In particular
embodiments, compositions of the invention comprise a surfactant,
e.g., about 0.001% (w/v) to about 0.05% (w/v) surfactant.
[0266] Examples of compositions of the invention include the
compositions described in Table 3, as well as these compositions
absent the surfactant.
[0267] In one embodiment, a composition comprises Antagonist A or a
modified form thereof at a concentration of about 3 mg/mL,
bevacizumab at a concentration of about 12.5 mg/mL, sodium
phosphate at a concentration of about 50 mM, sodium chloride at a
concentration of about 130 mM and polysorbate 20 at a concentration
of about 0.02% (w/v), wherein the pH of the composition is about
6.0.
[0268] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A or modified form thereof, about 12.5
mg/mL bevacizumab, about 50 mM sodium phosphate, about 5% (w/v)
sorbitol, and about 0.02% (w/v) polysorbate 20, wherein the pH of
the composition is about pH 6.0.
[0269] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A, or modified form thereof, about 12.5
mg/mL bevacizumab, about 50 mM sodium phosphate, about 5% (w/v)
sorbitol, and about 0.02% (w/v) polysorbate 20, wherein the pH of
the composition is about pH 7.0.
[0270] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A, or modified form thereof, about 12.5
mg/mL bevacizumab, about 50 mM sodium phosphate, about 150 mM NaCl,
and about 0.02% (w/v) polysorbate 20, wherein the pH of the
composition is about pH 7.0.
[0271] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A or modified form thereof, about 12.5
mg/mL bevacizumab, about 50 mM Tris.HCl, about 130 mM NaCl, and
about 0.02% (w/v) polysorbate 20, wherein the pH of the composition
is about pH 8.0.
[0272] In one embodiment, a composition of the invention comprises
about 15 mg/mL Antagonist A, or modified form thereof, about 12.5
mg/mL bevacizumab, about 30 mM sodium phosphate, about 75 mM NaCl,
about 3% (w/v) trehalose, and about 0.02% (w/v) polysorbate 20,
wherein the pH of the composition is about pH 6.3.
[0273] In one embodiment, a composition of the invention comprises
about 3 mg/mL Antagonist A, or modified form thereof, about 12.5
mg/mL bevacizumab or a pharmaceutically acceptable salt thereof,
about 30 mM sodium phosphate, about 75 mM NaCl, about 3% (w/v)
trehalose, and about 0.02% (w/v) polysorbate 20, wherein the pH of
the composition is about pH 6.3.
[0274] In particular embodiments, a composition comprising
Antagonist A or a modified form thereof and bevacizumab is
chemically stable for at least four weeks or at least eight weeks
at 25.degree. C. or for at least twelve weeks or at least 24 weeks
at 4.degree. C. In particular embodiments, at least 70% of each of
Antagonist A or modified form thereof and bevacizumab show no sign
of decomposition or modification resulting in formation of a new
chemical entity under these conditions.
[0275] Compositions Comprising Antagonist A and Aflibercept
[0276] In certain embodiments, a composition comprises Antagonist A
or a modified form thereof and aflibercept. In particular
embodiments, the ratio of the concentration (mass of Antagonist A
less that of its --R group/volume of composition) of Antagonist A
to the concentration (mass/volume of composition) of aflibercept
present in the composition is less than 25.0, less than 10.0, less
than 9.0, less than 8.0, less than 7.0, less than 6.0, less than
5.0, less than 4.0, less than 3.0, less than 2.0, less than 1.0,
less than 0.5, or less than 0.25.
[0277] Antagonist A's --R group is depicted in FIG. 78A.
[0278] In particular embodiments, a composition comprises
Antagonist A or a modified form thereof and aflibercept, and the
composition is stable with respect to both active agents at a
particular pH or suitable for parenteral administration. In certain
embodiments, the composition comprises one or more tonicity
modifier, surfactant, and buffer suitable to achieve or maintain
the particular pH or be suitable for parenteral administration.
Appropriate buffers include those described herein as well as
others known in the art, such as, e.g., a Good's buffers, e.g.,
MES.
[0279] In certain embodiments, the concentration of Antagonist A or
modified form thereof in the composition is less than about 50
mg/mL, less than about 40 mg/mL, less than about 30 mg/mL, less
than about 25 mg/mL, less than about 20 mg/mL, less than about 15
mg/mL, less than about 10 mg/mL, or less than about 5 mg/mL. In
certain embodiments, the concentration of Antagonist A or modified
form thereof is about 0.3 mg/mL to about 50 mg/mL, about 0.3 mg/mL
to about 40 mg/mL, about 0.3 mg/mL to about 30 mg/mL, about 0.3 to
about 25 mg/mL, about 0.3 mg/mL to about 20 mg/mL, about 0.3 mg/mL
to about 15 mg/mL, about 0.3 mg/mL to about 10 mg/mL, about 1 mg/mL
to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL
to about 30 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL
to about 20 mg/mL, about 1 mg/mL to about 15 mg/mL, about 1 mg/mL
to about 10 mg/mL, or about 1 mg/mL to about 5 mg/mL. In certain
embodiments, the concentration of Antagonist A or modified form
thereof is about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4
mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL,
about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL,
about 25 mg/mL, about 30 mg/mL, about 40 mg/mL, or about 50
mg/mL.
[0280] In certain embodiments, the concentration of aflibercept is
about 5 mg/mL to about 100 mg/mL, about 5 mg/mL to about 50 mg/mL,
about 5 mg/mL to about 40 mg/mL, about 10 mg/mL to about 100 mg/mL,
about 10 mg/mL to about 50 mg/mL, about 10 mg/mL to about 40 mg/mL,
about 20 mg/mL to about 40 mg/mL, about 30 mg/mL, about 50 mg/mL,
or about 40 mg/mL.
[0281] In certain embodiments, the concentration of aflibercept is
about 5 mg/mL to about 100 mg/mL, about 5 mg/mL to about 50 mg/mL,
about 5 mg/mL to about 40 mg/mL, about 10 mg/mL to about 100 mg/mL,
about 10 mg/mL to about 50 mg/mL, about 10 mg/mL to about 40 mg/mL,
about 20 mg/mL to about 40 mg/mL, about 30 mg/mL, about 50 mg/mL,
or about 40 mg/mL, and the concentration of Antagonist A or
modified form thereof is less than about 50 mg/mL, less than about
40 mg/mL, less than about 30 mg/mL, less than about 25 mg/mL, less
than about 20 mg/mL, less than about 15 mg/mL, less than about 10
mg/mL, or less than about 5 mg/mL.
[0282] In certain embodiments, the concentration of aflibercept is
about 5 mg/mL to about 100 mg/mL, about 5 mg/mL to about 50 mg/mL,
about 5 mg/mL to about 40 mg/mL, about 10 mg/mL to about 100 mg/mL,
about 10 mg/mL to about 50 mg/mL, about 10 mg/mL to about 40 mg/mL,
about 20 mg/mL to about 40 mg/mL, about 30 mg/mL, about 50 mg/mL,
or about 40 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1
mg/mL to about 5 mg/mL, and the concentration of Antagonist A or
modified form thereof is about 0.3 mg/mL to about 50 mg/mL, about
0.3 mg/mL to about 40 mg/mL, about 0.3 mg/mL to about 30 mg/mL,
about 0.3 to about 25 mg/mL, about 0.3 mg/mL to about 20 mg/mL,
about 0.3 mg/mL to about 15 mg/mL, about 0.3 mg/mL to about 10
mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40
mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 25
mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 15
mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 5
mg/mL.
[0283] In certain embodiments, the concentration of aflibercept is
about 5 mg/mL to about 100 mg/mL, about 5 mg/mL to about 50 mg/mL,
about 5 mg/mL to about 40 mg/mL, about 10 mg/mL to about 100 mg/mL,
about 10 mg/mL to about 50 mg/mL, about 10 mg/mL to about 40 mg/mL,
about 20 mg/mL to about 40 mg/mL, about 30 mg/mL, about 50 mg/mL,
or about 40 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1
mg/mL to about 5 mg/mL, and the concentration of Antagonist A or
modified form thereof is about 1 mg/mL, about 2 mg/mL, about 3
mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL,
about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about
20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 40 mg/mL, or about
50 mg/mL. In one embodiment, the concentration of Antagonist A is
about 3 mg/mL, and the concentration of aflibercept is about 20
mg/mL. In one embodiment, the concentration of Antagonist A is
about 6 mg/mL, and the concentration of aflibercept is about 40
mg/mL. In another embodiment, the concentration of Antagonist A or
modified form thereof is about 12 mg/mL, and the concentration of
alfibercept is about 80 mg/mL.
[0284] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and aflibercept, the
composition further comprises one or more tonicity modifier(s)
selected from sorbitol and sodium chloride. In particular
embodiments, the tonicity modifier comprises sorbitol, and the pH
of the composition is about 6.0 to about 8.0. In particular
embodiments, the tonicity modifier comprises sodium chloride, and
the pH of the composition is about 6.0 to about 8.0. In certain
embodiments, the tonicity modifier comprises sorbitol at about 1%
to about 10% (w/v), or about 1% (w/v), about 2% (w/v), about 3%
(w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7%
(w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v). In
particular embodiments, the tonicity modifier is sodium chloride at
a concentration of about 10 mM to about 200 mM, about 50 mM to 200
mM, about 75 mM to about 200 mM, about 25 mM to about 150 mM, about
50 mM to about 150 mM, about 20 mM, about 30 mM, about 40 mM, about
50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about
100 mM, about 110 mM, about 120 mM, about 130 mM about 140 mM or
about 150 mM. In one embodiment, the tonicity modifier is sodium
chloride at a concentration of about 40 mM.
[0285] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and aflibercept, the
composition further comprises a buffer capable of achieving or
maintaining the pH within a desired range. In certain embodiments,
the composition comprises one or more buffer(s) selected from
acetate, phosphate, histidine and Tris. In certain embodiments, the
buffer comprises phosphate at a concentration of about 1 mM to
about 200 mM, about 1 mM to about 50 mM, about 5 mM to about 200
mM, about 5 mM to about 100 mM, about 5 mM to about 50 mM, about 10
mM to about 150 mM, about 10 mM to about 100 mM, about 5 mM, about
10 mM, about 25 mM, or about 50 mM. In certain embodiments, the
phosphate buffer is sodium phosphate or potassium phosphate. In
particular embodiments, the pH of the buffered composition is about
5.0 to about 8.0, about 6.0 to about 8.0, about 5.5 to about 7.0,
about 6.0, about 7.0, or about 8.0. In one embodiment, the buffer
comprises phosphate, and the buffered composition has a pH of about
6.0 to about 8.0. In certain embodiments, the buffer comprises
phosphate at a concentration of about 5 mM to about 200 mM, about 5
mM to about 150 mM, about 5 mM to about 100 mM, about 5 mM, about
10 mM, about 25 mM, or about 50 mM, and the buffered composition
has a pH of about 5.5 to about 7.0, or about 6.0. In one particular
embodiment, the buffer comprises phosphate at a concentration of
about 10 mM, and the buffered composition has a pH of about
6.2.
[0286] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and aflibercept, the
composition further comprises sucrose. In particular embodiments,
sucrose is present in the composition at a concentration of about
0% (w/v) to about 10% (w/v), about 1% (w/v) to about 10% (w/v),
about 2% (w/v) to about 10% (w/v), or about 5% (w/v).
[0287] In certain embodiments of a composition comprising
Antagonist A or a modified form thereof and aflibercept, the
composition further comprises a surfactant. In particular
embodiments, the surfactant is polysorbate 20 at a concentration of
about 0.005% (w/v) to about 0.05% (w/v), about 0.01% (w/v) to about
0.05% (w/v), about 0.03% (w/v), or about 0.02% (w/v).
[0288] In one embodiment, a composition comprising Antagonist A or
a modified form thereof and aflibercept comprises Antagonist A or
modified form thereof, aflibercept, sodium chloride, and phosphate.
The composition may further comprise polysorbate or sucrose (or
both).
[0289] In one particular embodiment a composition comprises
Antagonist A or a modified form thereof and aflibercept; the ratio
of the concentration of Antagonist A or modified form thereof to
the concentration of aflibercept is less than 1; and the
composition further comprises sodium chloride at a concentration of
about 10 mM to about 200 mM, phosphate at a concentration of about
5 mM to about 50 mM, sucrose at a concentration of about 0% (w/v)
to about 10% (w/v), and polysorbate (e.g., polysorbate 20) at a
concentration of about 0.005% to about 0.05%, wherein the pH of the
composition is about 6.0 to about 8.0.
[0290] In certain embodiments, the compositions comprise: (a) about
0.3 mg/mL to about 30 mg/mL Antagonist A or a modified form
thereof, or pharmaceutically acceptable salt thereof; and (b) about
5 mg/mL to about 40 mg/mL aflibercept or pharmaceutically
acceptable salt thereof. In particular embodiments, the
compositions further comprise one or both of (c) about 5 mM to
about 50 mM phosphate buffer (e.g., about 5 mM to about 50 mM
sodium phosphate); and (d) about 10 mM to about 200 mM NaCl. In
further embodiments, the compositions further comprise: (e) 0 to
about 10% (w/v) sucrose. In certain embodiments, the compositions
comprise: (a) about 0.3 mg/mL to about 30 mg/mL Antagonist A or
modified form thereof, or pharmaceutically acceptable salt thereof;
(b) about 5 mg/mL to about 40 mg/mL aflibercept or pharmaceutically
acceptable salt thereof; (c) about 5 mM to about 50 mM phosphate
buffer; (d) about 10 mM to about 200 mM NaCl; and (e) 0 to about
10% (w/v) sucrose, wherein the pH of the composition is about pH
6.0 to about pH 8.0. In another embodiment, the compositions
further comprise: (f) about 0.001% (w/v) to about 0.05% (w/v)
polysorbate. In one particular embodiment, the compositions
comprise: (a) about 6 mg/mL Antagonist A or modified form thereof
or pharmaceutically acceptable salt thereof; (b) about 40 mg/mL
aflibercept or pharmaceutically acceptable salt thereof; (c) about
10 mM phosphate buffer, (d) about 40 mM NaCl; and (e) about 5%
(w/v) sucrose, wherein the pH of the composition is about pH 6.2.
In a further embodiment, the compositions further comprise: (f)
about 0.03% (w/v) polysorbate 20.
[0291] In certain embodiments of a composition of the invention
comprises: (a) about 0.3 mg/mL to about 30 mg/mL Antagonist A, or a
modified form thereof; (b) about 5 mg/mL to about 40 mg/mL
aflibercept; and one or more of (c) a buffer capable of achieving
or maintaining the pH of the composition to about pH 5.0 to about
pH 8.0; (d) a tonicity modifier; and (e) 0 to about 10% (w/v)
sucrose. In particular embodiments, the buffer, where present, is
about 5 mM to about 50 mM phosphate, and the tonicity modifier,
where present, is about 10 mM to about 200 mM NaCl.
[0292] In particular embodiments, a composition of the invention
comprises (a) about 0.3 mg/mL to about 30 mg/mL Antagonist A, or a
modified form thereof; (b) about 5 mg/mL to about 40 mg/mL
aflibercept; (c) about 5 mM to about 50 mM phosphate; (d) about 10
mM to about 200 mM NaCl; (e) 0 to about 10% (w/v) sucrose; and (f)
about 0.001% (w/v) to about 0.05% (w/v) surfactant, wherein the pH
of the composition is about pH 6.0 to about pH 8.0.
[0293] Compositions of the invention also include any of the
compositions described herein absent the surfactant.
[0294] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof at a concentration of about
6 mg/mL, aflibercept at a concentration of about 40 mg/mL,
phosphate at a concentration of about 10 mM, sodium chloride at a
concentration of about 40 mM and polysorbate 20 at a concentration
of about 0.03% (w/v), and the composition has a pH about 6.2.
[0295] In another embodiment, a composition of the invention
comprises Antagonist A or a modified form thereof at a
concentration of about 3 mg/mL, aflibercept at a concentration of
about 20 mg/mL, phosphate at a concentration of about 10 mM, sodium
chloride at a concentration of about 40 mM and polysorbate 20 at a
concentration of about 0.03% (w/v), and the composition has a pH
about 6.2.
[0296] In particular embodiments, a composition comprising
Antagonist A and aflibercept is chemically stable for at least four
weeks or at least eight weeks at 25.degree. C. or for at least
twelve weeks or at least 24 weeks at 4.degree. C. In particular
embodiments, at least 70% of both antagonists show no sign of
decomposition or modification resulting in formation of a new
chemical entity under these conditions.
[0297] Methods for Making Compositions of the Invention
[0298] Compositions of the invention, including those described
herein, may be prepared by a method comprising, consisting
essentially of, or consisting of, admixing the antagonists (e.g.,
one or more anti-PDGF aptamers and one or more VEGF antagonists)
and an effective amount of a buffer, e.g., a histidine, phosphate,
acetate or Tris buffer, and optionally adjusting the pH of the
resulting mixture to a pH of about 5.5 to about 8.0 and variations
in between as described herein.
[0299] In some embodiments, the method further comprises, consists
essentially of, or consists of admixing the anti-PDGF aptamer and
the VEGF antagonist and an effective amount of a tonicity agent. In
a particular aspect, the tonicity agent is sodium chloride or
sorbitol.
[0300] In some embodiments, the method further comprises, consists
essentially of, or consists of admixing the anti-PDGF aptamer and
VEGF antagonist and an effective amount of a surfactant. In
particular aspects, the surfactant is a polysorbate, e.g., Tween 20
or Tween 80.
[0301] In some embodiments, the method further comprises, consists
essentially of, or consists of admixing the anti-PDGF aptamer and
VEGF antagonist and an effective amount of a stabilizer,
cryoprotectant, or lyoprotectant. The stabilizer can be at least
one a sugar, an amino acid, a polyol, a surfactant, an antioxidant,
a preservative, a cyclodextrin, a polyethyleneglycol, albumin or a
salt.
[0302] In particular aspects of the method, the compositions are
prepared by admixing the anti-PDGF aptamer and the VEGF antagonist
and various excipients present in the various compositions
described herein and in the range of concentrations described
herein, including each the specific compositions described above
that comprise Antagonist A or a modified form thereof in
combination with either bevacizumab, ranibizumab, or
aflibercept.
[0303] Thus, in one embodiment, a composition of the invention is
prepared by admixing the following: Antagonist A or a modified form
thereof to a final concentration of about 3 mg/mL, bevacizumab to a
final concentration of about 12.5 mg/mL, phosphate to a final
concentration of about 50 mM, sodium chloride to a final
concentration of about 130 mM, and polysorbate 20 to a final
concentration of about 0.02% (w/v). In another embodiment, a
composition of the invention is prepared by admixing the following:
Antagonist A or a modified form thereof to a final concentration of
about 6 mg/mL, bevacizumab to a final concentration of about 25
mg/mL, phosphate to a final concentration of about 50 mM, sodium
chloride to a final concentration of about 130 mM, and polysorbate
20 to a final concentration of about 0.02% (w/v). In certain
embodiments, the pH of the composition is adjusted to about
6.0.
[0304] In another embodiment, a composition is prepared by admixing
the following: Antagonist A or a modified form thereof to a final
concentration of about 3 mg/mL, ranibizumab to a final
concentration of about 5 mg/mL, histidine to a final concentration
of about 10 mM, sodium chloride to a final concentration of about
130 mM and polysorbate 20 to a final concentration of about 0.02%
(w/v). In another embodiment, a composition is prepared by admixing
the following: Antagonist A or a modified form thereof to a final
concentration of about 6 mg/mL, ranibizumab to a final
concentration of about 10 mg/mL, histidine to a final concentration
of about 10 mM, sodium chloride to a final concentration of about
130 mM and polysorbate 20 to a final concentration of about 0.02%
(w/v). In certain embodiments, the pH of the composition is
adjusted to about 6.0.
[0305] In another embodiment, a composition is prepared by admixing
the following: Antagonist A or a modified form thereof to a final
concentration of about 6 mg/mL, aflibercept to a final
concentration of about 40 mg/mL, phosphate to a final concentration
of about 10 mM, sodium chloride to a final concentration of about
40 mM, sucrose to a final concentration of about 5% (w/v) and
polysorbate 20 to a final concentration of about 0.03% (w/v). In
another embodiment, a composition is prepared by admixing the
following: Antagonist A or a modified form thereof to a final
concentration of about 3 mg/mL, aflibercept to a final
concentration of about 20 mg/mL, phosphate to a final concentration
of about 10 mM, sodium chloride to a final concentration of about
40 mM, sucrose to a final concentration of about 5% (w/v) and
polysorbate 20 to a final concentration of about 0.03% (w/v). In
certain embodiments, the pH of the composition is adjusted to about
6.2.
[0306] In certain embodiments, the compositions are admixed in a
glass vial or syringe or are stored after admixing in a glass
viable or a syringe.
Methods of Treating or Preventing Opthalmological Diseases
[0307] Compositions of the invention are useful for treating or
preventing a variety of ophthalmological diseases. In some
embodiments, the ophthalmological disease is a neovascular
disorder. In other embodiments, the ophthalmological disease
results in retinal edema. Illustrative ophthalmological diseases
that can be treated or prevented by the present invention are
described herein.
[0308] In certain embodiments, the invention provides methods for
treating or preventing an ophthalmological disease, comprising
administering to a mammal in need thereof a composition of the
invention. In particular embodiments, an anti-PDGF aptamer present
in the composition is Antagonist A or a modified form thereof. In
particular embodiments, a VEGF antagonist present in the
composition is ranibizumab, bevacizumab, or aflibercept. In
particular embodiments, therapeutic agents present in compositions
of the invention comprise an effective amount of: (i) Antagonist A
or a modified form thereof and ranibizumab; (ii) a Antagonist A or
a modified form thereof and bevacizumab; or (iii) Antagonist A or a
modified form thereof and aflibercept.
[0309] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof, ranibizumab, histidine,
and sodium chloride. The composition may further comprise
polysorbate.
[0310] In one particular embodiment, the composition of the
invention comprises Antagonist A or modified form thereof and
ranibizumab at a ratio of the concentration of Antagonist A or
modified form thereof to the concentration of bevacizumab of less
than 2, sodium chloride at a concentration of about 10 mM to about
200 mM, histidine at a concentration of about 1 mM to about 100 mM,
and polysorbate (e.g., polysorbate 20) at a concentration of about
0.005% to about 0.05% or 0.001% to about 0.05%, wherein the pH of
the composition is about 5.5 to about 7.0.
[0311] In one embodiment, the composition of the invention
comprises Antagonist A or a modified form thereof at a
concentration of about 3 mg/mL, ranibizumab at a concentration of
about 5 mg/mL, histidine at a concentration of about 10 mM, sodium
chloride at a concentration of about 130 mM and polysorbate 20 at a
concentration of about 0.02% (w/v), wherein the pH of the
composition is about 6.0. In a further embodiment, the composition
comprises Antagonist A or a modified form thereof at a
concentration of about 6 mg/mL, ranibizumab at a concentration of
about 10 mg/mL, histidine at a concentration of about 10 mM, sodium
chloride at a concentration of about 130 mM and polysorbate 20 at a
concentration of about 0.02% (w/v), wherein the pH of the
composition is about 6.0.
[0312] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof, bevacizumab, sodium
chloride, phosphate, and polysorbate. The composition may further
comprise polysorbate.
[0313] In one particular embodiment, the composition of the
invention comprises Antagonist A or modified form thereof and
bevacizumab at a ratio of the concentration of Antagonist A or
modified form thereof to the concentration of bevacizumab of less
than 1, sodium chloride at a concentration of about 10 mM to about
200 mM, phosphate at a concentration of about 5 mM to about 200 mM,
and polysorbate (e.g., polysorbate 20) at a concentration of about
0.005% to about 0.05%, wherein the pH of the composition is about
5.5 to about 7.0.
[0314] In one embodiment, the composition of the invention
comprises Antagonist A or a modified form thereof at a
concentration of about 3 mg/mL, bevacizumab at a concentration of
about 12.5 mg/mL, phosphate at a concentration of about 50 mM,
sodium chloride at a concentration of about 130 mM and polysorbate
20 at a concentration of about 0.02% (w/v), wherein the pH of the
composition is about 6.0. In another embodiment, the composition
comprises Antagonist A or a modified form thereof at a
concentration of about 6 mg/mL, bevacizumab at a concentration of
about 25 mg/mL, phosphate at a concentration of about 50 mM, sodium
chloride at a concentration of about 130 mM and polysorbate 20 at a
concentration of about 0.02% (w/v), wherein the pH of the
composition is about 6.0.
[0315] In one embodiment, a composition of the invention comprises
Antagonist A or a modified form thereof and aflibercept, sodium
chloride, and phosphate. The composition may further comprise
polysorbate or sucrose (or both).
[0316] In one particular embodiment, the composition of the
invention comprises Antagonist A or a modified form thereof and
aflibercept at a ratio of the concentration of Antagonist A to the
concentration of aflibercept of less than 1, sodium chloride at a
concentration of about 10 mM to about 200 mM, phosphate at a
concentration of about 5 mM to about 50 mM, sucrose at a
concentration of about 0% (w/v) to about 10% (w/v), and polysorbate
(e.g., polysorbate 20) at a concentration of about 0.005% to about
0.05%, wherein the composition has a pH of about 6.0 to about
8.0.
[0317] In one embodiment, the composition of the invention
comprises Antagonist A or a modified form thereof at a
concentration of about 6 mg/mL, aflibercept at a concentration of
about 40 mg/mL, phosphate at a concentration of about 10 mM, sodium
chloride at a concentration of about 40 mM and polysorbate 20 at a
concentration of about 0.03% (w/v), wherein the composition has a
pH of about 6.2.
[0318] Ophthalmological Diseases
[0319] In certain embodiments, the ophthalmological disease is
age-related macular degeneration. Examples of age-related macular
degeneration are nonneovascular (also known as "Dry") and
neovascular (also known as "Wet") macular degeneration. In one
embodiment, the dry age-related macular degeneration is associated
with the formation of drusen. In some embodiments, treating or
preventing dry macular degeneration encompasses treating or
preventing an abnormality of the retinal pigment epithelium.
Examples of abnormalities of the retinal pigment epithelium include
geographic atrophy, non-geographic atrophy, focal hypopigmentation,
and focal hyperpigmentation. In some embodiments, treating or
preventing wet age-related macular degeneration encompasses
treating or preventing choroidal neovascularization or pigment
epithelial detachment.
[0320] In other embodiments, the ophthalmological disease is
polypoidal choroidal vasculopathy. Polypoidal choroidal
vasculopathy is characterized by a lesion from an inner choroidal
vascular network of vessels ending in an aneurysmal bulge or
outward projection (Ciardella et al (2004) Surv Ophthalmol 49
25-37).
[0321] In certain embodiments, the ophthalmological disease is a
condition associated with choroidal neovascularization. Examples of
conditions associated with choroidal neovascularization include a
degenerative, inflammatory, traumatic or idiopathic condition. In
some embodiments, treating or preventing a degenerative disorder
associated with choroidal neovascularization encompasses treating
or preventing a heredodegenerative disorder. Examples of
heredodegenerative disorders include vitelliform macular dystrophy,
fundus flavimaculatus and optic nerve head drusen. Examples of
degenerative conditions associated with choroidal
neovascularization include myopic degeneration or angioid streaks.
In other embodiments, treating or preventing an inflammatory
disorder associated with choroidal neovascularization encompasses
treating or preventing ocular histoplasmosis syndrome, multifocal
choroiditis, serpimnous choroiditis, toxoplasmosis, toxocariasis,
rubella, Vogt-Koyanagi-Harada syndrome. Behcet syndrome or
sympathetic ophthalmia. In still other embodiments, treating or
preventing a traumatic disorder associated with choroidal
neovascularization encompasses treating or preventing choroidal
rupture or a traumatic condition caused by intense
photocoagulation.
[0322] In other embodiments, the ophthalmological disease is
hypertensive retinopathy or sicle cell retinopathy.
[0323] In one embodiment, the ophthalmological disease is diabetic
retinopathy. Diabetic retinopathy can be nonproliferative or
proliferative diabetic retinopathy. Examples of nonproliferative
diabetic retinopathy include macular edema and macular
ischemia.
[0324] In particular embodiments, the ophthalmological disease is a
condition associated with peripheral retinal neovascularization.
Examples of conditions associated with peripheral retinal
neovascularization include ischemic vascular disease, inflammatory
disease with possible ischemia, incontinentia pigmenti, retinitis
pigmentosa, retinoschisis or chronic retinal detachment. Examples
of ischemic vascular disease include proliferative diabetic
retinopathy, branch retinal vein occlusion, branch retinal
arteriolar occlusion, carotid cavernous fistula, sickling
hemoglobinopathy, non-sickling hemoglobinopathy, IRVAN syndrome
(retinal vasculitic disorder characterized by idiopathic retinal
vasculitis, an aneurysm, and neuroretinitis), retinal embolization,
retinopathy of prematurity, familial exudative vitreoretinopathy,
hyperviscosity syndrome, aortic arch syndrome or Eales disease.
Examples of sickling hemoglobinopathy include SS hemoglobinopathy
and SC hemoglobinopathy. Examples of non-sickling hemoglobinopathy
include AC hemoglobinopathy and AS hemoglobinopathy. Examples of
hyperviscosity syndrome include leukemia, Waldenstrom
macroglobulinemia, multiple myeloma, polycythemia or
myeloproliferative disorder.
[0325] In some embodiments, treating or preventing an inflammatory
disease with possible ischemia encompasses treating or preventing
retinal vasculitis associated with systemic disease, retinal
vasculitis associated with an infectious agent, uveitis or birdshot
retinopathy. Examples of systemic diseases include systemic lupus
erythematosis. Behcet's disease, inflammatory bowel disease,
sarcoidosis, multiple sclerosis, Wegener's granulomatosis and
polyarteritis nodosa. Examples of infectious agents include a
bacterial agent that is the causative agent for syphilis,
tuberculosis, Lyme disease or cat-scratch disease, a virus such as
herpesvirus, or a parasite such as Toxocara canis or Toxoplasma
gondii. Examples of uveitis include pars planitis or Fuchs uveitis
syndrome.
[0326] In certain embodiments, the ophthalmological disease is
retinopathy of prematurity. Retinopathy of prematurity can result
from abnormal growth of blood vessels in the vascular bed
supporting the developing retina (Pollan C (2009) Neonatal Netw.
28:93-101).
[0327] In other embodiments, the ophthalmological disease is venous
occlusive disease or arterial occlusive disease. Examples of venous
occlusive disease include branch retinal vein occlusion and central
retinal vein occlusion. A branch retinal vein occlusion can be a
blockage of the portion of the circulation that drains the retina
of blood. The blockage can cause back-up pressure in the
capillaries, which can lead to hemorrhages and also to leakage of
fluid and other constituents of blood. Examples of arterial
occlusive disease include branch retinal artery occlusion, central
retinal artery occlusion or ocular ischemic syndrome. A branch
retinal artery occlusion (BRAO) can occur when one of the branches
of the arterial supply to the retina becomes occluded.
[0328] In particular embodiments, the ophthalmological disease is
central serous chorioretinopathy (CSC). In one embodiment, CSC is
characterized by leakage of fluid in the central macula
[0329] In one embodiment, the ophthalmological disease is cystoid
macular edema (CME) In certain embodiments, CME affects the central
retina or macula. In another embodiment, CME occurs after cataract
surgery.
[0330] In other embodiments, the ophthalmological disease is
retinal telangiectasia. In one embodiment, retinal telangiectasia
is characterized by dilation and tortuosity of retinal vessels and
formation of multiple aneurysms. Idiopathic JXT, Leber's miliary
aneurysms, and Coats' disease are three types of retinal
telangiectasias
[0331] In one embodiment, the ophthalmological disease is arterial
macroaneurysm.
[0332] In one embodiment, the ophthalmological disease is retinal
angiomatosis. In one embodiment, retinal angiomatosis occurs when
the ocular vessels form multiple angiomas
[0333] In one embodiment, the ophthalmological disease is
radiation-induced retinopathy (RIRP). In one embodiment. RIRP may
display symptoms such as macular edema and nonproliferative and
proliferative retinopathy
[0334] In certain embodiments, the ophthalmological disease is
rubeosis iridis. In one embodiment, rubeosis iridis results in the
formation of neovascular glaucoma. In another embodiment, rubeosis
iridis is caused by diabetic retinopathy, central retinal vein
occlusion, ocular ischemic syndrome, or chronic retinal
detachment.
[0335] In certain embodiments, the ophthalmological disease is a
neoplasm. Examples of neoplams include an eyelid tumor, a
conjunctival tumor, a choroidal tumor, an iris tumor, an optic
nerve tumor, a retinal tumor, an infiltrative intraocular tumor or
an orbital tumor. Examples of an eyelid tumor include basal cell
carcinoma, squamous carcinoma, sebaceous carcinoma, malignant
melanoma, capillary hemangioma, hydrocystoma, nevus or seborrheic
keratosis. Examples of a conjunctival tumor include conjunctival
Kaposi's sarcoma, squamous carcinoma, intraepithelial neoplasia of
the conjunctiva, epibular dermoid, lymphoma of the conjunctiva,
melanoma, pingueculum, or pterygium. Examples of a choroidal tumor
include choroidal nevus, choroidal hemangioma, metastatic choroidal
tumor, choroidal osteoma, choroidal melanoma, ciliary body melanoma
or nevus of Ota. Examples of an iris tumor include anterior uveal
metastasis, iris cyst, iris melanocytoma, iris melanoma, or pearl
cyst of the iris. Examples of an optic nerve tumor include optic
nerve melanocytoma, optic nerve sheath meningioma, choroidal
melanoma affecting the optic nerve, or circumpapillary metastasis
with optic neuropathy. Examples of a retinal tumor include retinal
pigment epithelial (RPE) hypertrophy, RPE adenoma, RPE carcinoma,
retinoblastoma, hamartoma of the RPE, or von Hippel angioma.
Examples of an infiltrative intraocular tumor include chronic
lymphocytic leukemia, infiltrative choroidopathy, or intraocular
lymphoma. Examples of an orbital tumor include adenoid cystic
carcinoma of the lacrimal gland, cavernous hemangioma of the orbit,
lymphangioma of the orbit, orbital mucocele, orbital pseudotumor,
orbital rhabdomyosarcoma, periocular hemangioma of childhood, or
sclerosing orbital pseudotumor.
[0336] The compositions of the invention can be administered alone
or in conjunction with another therapy and can be provided at home,
a doctor's office, a clinic, a hospital's outpatient department, or
a hospital. The duration of the administration can depend on the
type of ophthalmological disease being treated or prevented, the
age and condition of the mammal, the stage and type of the mammal's
disease, and how the mammal responds to the treatment. In
particular embodiments, the mammal is a human. Additionally, a
mammal having a greater risk of developing an ophthalmological
disease (e.g., a diabetic patient) can receive treatment to inhibit
or delay the onset of symptoms. In one embodiment, the present
methods or compositions allow for the administration of a
relatively lower dose of one or more of the anti-PDGF aptamer(s)
and VEGF antagonist(s) present in the composition, as compared to
the dose utilized when the therapeutic agent is used alone.
[0337] Administration of the composition of the invention may be by
any suitable means that results in an amount of anti-PDGF aptamer
and VEGF antagonist that is effective for the treatment or
prevention of an ophthalmological disease. In one embodiment, the
composition is administered in an amount effective to treat or
prevent an ophthalmological disease.
[0338] The dosage of composition administered can depend on several
factors including the severity of the condition, whether the
condition is to be treated or prevented, and the age, weight, and
health of the person to be treated. Additionally, pharmacogenomic
(the effect of genotype on the pharmacokinetic, pharmacodynamic or
efficacy profile of a therapeutic) information about a particular
patient may affect dosage used. Furthermore, the exact individual
dosages can be adjusted somewhat depending on a variety of factors,
including the specific combination of therapeutic agents present in
the composition, the time of administration, the route of
administration, the nature of the composition, the rate of
excretion, the particular ophthalmological disease being treated,
the severity of the disorder, and the anatomical location of the
disorder. The amount of each antagonist that is admixed with the
carrier materials to produce a single dosage can vary depending
upon the mammal being treated and the particular mode of
administration.
[0339] For administration of compositions by parenteral injection,
the dosage of each of the anti-PDGF aptamer and VEGF antagonist is
typically 0.1 mg to 250 mg per day, 1 mg to 20 mg per day, or 3 mg
to 5 mg per day. Injections may be given up to four times daily.
Generally, when parenterally administered, the dosage of an
anti-PDGF aptamer or VEGF antagonist for use in the present
invention is typically 0.1 mg to 1500 mg per day, or 0.5 mg to 10
mg per day, or 0.5 mg to 5 mg per day. A dosage of at least up to
3000 mg per day can be administered.
[0340] When ophthalmologically administered to a human, for example
intravitreally, the dosage of each of the anti-PDGF aptamer and
VEGF antagonist present in the composition of the invention is
typically 0.003 mg to 5.0 mg per eye per administration, or 0.03 mg
to 3.0 mg per eye per administration, or 0.1 mg to 1.0 mg per eye
per administration In one embodiment, the dosage of one or more
anti-PDGF aptamer in the composition is 0.03 mg, 0.3 mg, 1.5 mg or
3.0 mg per eye. In another embodiment, the dosage of VEGF
antagonist in the composition is about 0.5 mg per eye. The dosage
can range from 0.01 mL to 0.2 mL administered per eye, or 0.03 mL
to 0.15 mL administered per eye, or 0.05 mL to 0.10 mL administered
per eye. For example, in certain embodiments, the anti-PDGF aptamer
Antagonist A is delivered intravitreally at up to 30 mg/ml with
injection volumes up to 100 .mu.L.
[0341] Administration of the composition of the invention may be
one to four times daily or one to four times per month or one to
six times per year or once every two, three, four or five years.
Administration can be for the duration of one day or one month, two
months, three months, six months, one year, two years, three years,
and may even be for the life of the patient. In one embodiment, the
administration is performed once a month for three months. Chronic,
long-term administration will be indicated in many cases. The
dosage may be administered as a single dose or divided into
multiple doses. In general, the desired dosage should be
administered at set intervals for a prolonged period, usually at
least over several weeks or months, although longer periods of
administration of several months or years or more may be
needed.
[0342] In addition to treating pre-existing ophthalmological
diseases, the compositions can be administered prophylactically in
order to prevent or slow the onset of these disorders. In
prophylactic applications, the composition can be administered to a
mammal susceptible to or otherwise at risk of a particular
ophthalmological disease.
[0343] In one embodiment, the compositions of the invention are
administered to a mammal in need of treatment thereof, typically in
the form of an injectable pharmaceutical composition. The
administration can be by injection, for example by intraocular
injection, or by using a drug delivery device. Parenteral,
systemic, or transdermal administration is also within the scope of
the invention.
[0344] Compositions may be formulated to release the anti-PDGF
aptamer or VEGF antagonist substantially immediately upon
administration or at any predetermined time period after
administration, using controlled release compositions. For example,
a composition can be provided in sustained-release form. The use of
immediate or sustained-release compositions depends on the nature
of the condition being treated. For example, if the condition
consists of an acute disorder, treatment with an immediate release
form may be used over a prolonged release composition. For certain
preventative or long-term treatments, a sustained released
composition can also be used.
[0345] Many strategies can be pursued to obtain controlled release
in which the rate of release outweighs the rate of degradation or
metabolism of the therapeutic agents. For example, controlled
release can be obtained by the appropriate selection of composition
parameters and ingredients, including, e.g., appropriate controlled
release compositions and coatings. Examples include oil solutions,
suspensions, emulsions, microcapsules, microspheres, nanoparticles,
patches, and liposomes. Depot formulations may also be used. e.g.,
in the form of microparticles, implants, or solid boluses that form
in situ. Depot formulations may comprise a biodegradable polymer
excipient that controls the rate of drug release and resorb during
or after drug release. One class of biodegradable polymers is
lactide/glycolide polymers. These resorbable polymers are
biocompatible and are believed to resorb by hydrolysis, initially
to lactic acid and glycolic acid, and eventually to carbon dioxide
and water.
[0346] Compositions of the invention can also be delivered using a
drug-delivery device such as an implant. Such implants can be
biodegradable or biocompatible, or can be non-biodegradable. The
implants can be permeable to the anti-PDGF aptamer or VEGF
antagonist or deliver the agents by bioerosion. Ophthalmic drug
delivery devices can be inserted into a chamber of the eye, such as
the anterior or posterior chamber or can be implanted in or on the
sclera, choroidal space, or an avascularized region exterior to the
vitreous. In one embodiment, the implant can be positioned over an
avascular region, such as on the sclera, so as to allow for
transcleral diffusion of the anti-PDGF aptamer and VEGF antagonist
to the desired site of treatment, e.g., the intraocular space and
macula of the eye. Furthermore, the site of transcleral diffusion
can be proximal to a site of neovascularization such as a site
proximal to the macula. Suitable drug delivery devices are
described, for example, in U.S. Publication Nos. 2008/0286334;
2008/0145406; 2007/0184089; 2006/0233860; 2005/0244500;
2005/0244471; and 2005/0244462, and U.S. Pat. No. 6,808,719 and
5,322,691, the contents of each of which is herein incorporated by
reference in its entirety.
[0347] In one embodiment, the implant comprises a composition of
the invention dispersed in a biodegradable polymer matrix. The
matrix can comprise PLGA (polylactic acid-polyglycolic acid
copolymer), an ester-end capped polymer, an acid end-capped
polymer, or a mixture thereof. In another embodiment, the implant
comprises a composition comprising an anti-PDGF aptamer and a VEGF
antagonist, a surfactant, and lipophilic compound. The lipophilic
compound can be present in an amount of about 80-99% by weight of
the implant. Suitable lipophilic compounds include, but are not
limited to, glyceryl palmitostearate, diethylene glycol
monostearate, propylene glycol monostearate, glyceryl monostearate,
glyceryl monolinoleate, glyceryl monooleate, glyceryl
monopalmitate, glyceryl monolaurate, glyceryl dilaurate, glyceryl
monomyristate, glyceryl dimyristate, glyceryl monopalmitate,
glyceryl dipalmitate, glyceryl monostearate, glyceryl distearate,
glyceryl monooleate, glyceryl dioleate, glyceryl monolinoleate,
glyceryl dilinoleate, glyceryl monoarachidate, glyceryl
diarachidate, glyceryl monobehenate, glyceryl dibehenate, and
mixtures thereof.
[0348] In another embodiment, the implant comprises a composition
of the invention housed within a hollow sleeve. The composition
comprising the anti-PDGF aptamer and VEGF antagonist are delivered
to the eye by inserting the sleeve into the eye, releasing the
implant from the sleeve into the eye, and then removing the sleeve
from the eye. An example of this delivery device is described in
U.S. Publication No. 2005/0244462, which is hereby incorporated by
reference in its entirety.
[0349] In one embodiment, the implant is a flexible ocular insert
device adapted for the controlled sustained release of an anti-PDGF
aptamer and a VEGF antagonist into the eye. In one embodiment, the
device includes an elongated body of a polymeric material in the
form of a rod or tube containing a composition comprising an
anti-PDGF aptamer and a VEGF antagonist, and with at least two
anchoring protrusions extending radially outwardly from the body.
The device may have a length of at least 8 mm and the diameter of
its body portion including the protrusions does not exceed 1.9 mm.
The sustained release mechanism can, for example, be by diffusion
or by osmosis or bioerosion. The insert device can be inserted into
the upper or lower fornix of the eye so as to be independent of
movement of the eye by virtue of the fornix anatomy. The
protrusions can be of various shapes such as, for example, ribs,
screw threads, dimples or bumps, truncated cone-shaped segments or
winding braid segments. In a further embodiment, the polymeric
material for the body is selected as one which swells in a liquid
environment. Thus a device of smaller initial size can be employed.
The insert device can be of a size and configuration such that,
upon insertion into the upper or lower fornix, the device remains
out of the field of vision so as to be well retained in place and
imperceptible by a recipient over a prolonged period of use. The
device can be retained in the upper or lower fornix for 7 to 14
days or longer. An example of this device is described in U.S. Pat.
No. 5,322,691, which is hereby incorporated by reference in its
entirety.
[0350] In certain embodiments, compositions of the invention can
also be delivered using a drug-delivery device such as an exoplant,
e.g., an episcleral oxplant, such as one described in Pontes de
Carvalho, R. A. et al., Invest Ophthalmol Vis Sci. 2006,
47(1):4532-9, incorporated by reference in its entirety. Such
exoplants can be biodegradable or biocompatible, or can be
non-biodegradable.
[0351] In other embodiments, compositions of the invention can also
be delivered using a drug-delivery device such as a refillable
intraocular depot.
[0352] Dosing is generally dependent on severity and responsiveness
of the condition to be treated, with course of treatment lasting
from several days to several months or until a cure is effected or
a diminution of disease state is achieved. Optimal dosing schedules
can be calculated from measurements of drug accumulation in the
body or at a localized site or based upon a patient's response.
Persons of ordinary skill can optimize dosages, dosing
methodologies, and repetition rates. Optimum dosages may vary
depending on the potency of anti-PDGF agonists and VEGF
antagonists, and may also be estimated based on EC50's in vitro and
in vivo animal studies.
EXAMPLES
Example 1
Stability of Compositions Comprising Antagonist A and
Ranibizumab
[0353] The composition stability of Antagonist A and ranibizumab,
commercially available as Lucentis.RTM. from Genentech (S. San
Francisco, Calif.), in various compositions was examined under a
range of conditions. Various pHs (5.0-8.0) and tonicity modifiers
(sodium chloride, sorbitol, and trehalose) were used to optimize
the composition stability at various storage conditions (4.degree.
C., 25.degree. C., and 37.degree. C.) and under a physical stress
(agitation). The composition stability of Antagonist A and
ranibizumab was characterized by visual observation, pH
measurement, and various HPLC methods (anion exchange [AEX-HPLC],
weak cation exchange [WCX-HPLC], and size exclusion [SE-HPLC]).
[0354] Throughout the 16 weeks of the study, it was determined that
among the compositions examined a composition comprising Antagonist
A at 3 mg/mL and ranibizumab at 5 mg/mL in 10 mM L-histidine at pH
6.0, 130 mM NaCl, 0.01% (w/v) polysorbate 20 (F6) was the most
stable and provided the greatest protection against the degradation
of Antagonist A and ranibizumab. A more detailed description of the
experiments performed is provided herein.
Composition Parameters
[0355] The following composition parameters were examined: [0356]
(1) pH: 4.0, 5.0, 6.0, 6.5, 7.0, 7.3, 8.0 [0357] (2) Buffers:
Acetate, Phosphate, Histidine and [0358]
2-Amino-2-hydroxymethyl-propane-1,3-diol ("Tris") [0359] (3)
Tonicity Modifiers: Sodium Chloride. Sorbitol, and Trehalose [0360]
(4) Surfactants: Polysorbate 20 [0.01% and 0.005% (% w/v)]
[0361] The following parameters were fixed: [0362] (1) Fill volume
was 300 .mu.L in modified 3 cc vials provided by Ophthotech Corp.
(obtained from Mglas AG, Munnerstadt, Germany) [0363] (2) The
concentration of ranibizumab was 5 mg/mL [0364] (3) The
concentration of Antagonist A was fixed at 3 mg/mL
[0365] Table 1 below summarizes the composition matrix used in this
study.
TABLE-US-00005 TABLE 1 Composition Matrix [Ant. A] [ran.]
Polysorbate Comp. Buffer pH Tonicity Modifier (mg/mL) (mg/mL) 20 (%
w/v) F1 10 mM Sodium 7.3 150 mM NaCl 3 0 0% Phosphate F2 10 mM
Sodium 5.0 5% (w/v) Sorbitol 3 5 0.01% Acetate F3 10 mM Sodium 5.0
130 mM NaCl 3 5 0.01% Acetate F4 10 mM Histidine.cndot.HCl 5.5 10%
(w/v) 0 5 0.01% Trehalose F5 10 mM Histidine.cndot.HCl 6.0 5% (w/v)
Sorbitol 3 5 0.01% F6 10 mM Histidine.cndot.HCl 6.0 130 mM NaCl 3 5
0.01% F7 10 mM Sodium 7.0 5% (w/v) Sorbitol 3 5 0.01% Phosphate F8
10 mM Sodium 7.0 130 mM NaCl 3 5 0.01% Phosphate F9 10 mM
Tris.cndot.HCl 8.0 5% (w/v) Sorbitol 3 5 0.01% F10 10 mM
Tris.cndot.HCl 8.0 130 mM NaCl 3 5 0.01% F11 5 mM Sodium 6.5 75 mM
NaCl + 3 5 0.005% Phosphate + 5 mM 5% (w/v) Histidine Trehalose
"Ant. A" is Antagonist A; "ran." is ranibizumab
Sample Preparation
[0366] In order to obtain a 3 mg/mL concentration of Antagonist A
in the composition, an Antagonist A stock solution was prepared at
6 mg/mL in 10 mM phosphate, 150 mM NaCl, and pH 7.3. The resulting
stock solution was admixed 1:1 with a diluted form of commercial
Lucentis.RTM. (10 mg/mL), resulting in final concentrations of 3
mg/mL Antagonist A and 5 mg/mL ranibizumab (F11). The composition
was placed in 10 kDa molecular weight cutoff dialysis cassettes and
dialyzed .about.1,000,000-fold against the various composition
buffers listed in Table 1 (Comp. Nos. F2-F3, F5-F10).
Composition Studies
[0367] The compositions were tested under the following conditions
(although certain compositions were not tested at all time points
due to degradation at earlier time points):
TABLE-US-00006 TABLE 2 Test Conditions Conditions Timepoints
4.degree. C. 0, 2, 4, 8, 12, and 16 weeks 25.degree. C. 2, 4, 8,
and 12 weeks 37.degree. C. 2, 4, 8, and 12 weeks Agitation 4
hours
Analytical Methods
[0368] In order to measure the concentration of any degradation
products generated under stress in the various compositions, the
following stability-indicating assays were used:
[0369] (1) SE-HPLC (Analysis of Antagonist A and ranibizumab)
[0370] Mobile Phase: 50 mM phosphate buffer, 100 mM sodium
chloride, pH 7.0 [0371] Column: Tosoh TSKgel G3000SWXL 7.8
mm.times.300 mm, 5 .mu.m particles [0372] Column Temperature:
Ambient [0373] Flow Rate: 1.0 mL/min [0374] Wavelength: Signal, 280
nm; Reference. 360 nm [0375] Injection volume: 5 .mu.L [0376]
Sample Preparation: No dilution [0377] Percent purity reported
based on integrated area percent of main peaks identified for both
Antagonist A and ranibizumab.
[0378] (2) WCX-HPLC (Analysis of ranibizumab) [0379] Mobile Phase
A: 10 mM phosphate buffer, pH 7.0 [0380] Mobile Phase B: 10 mM
phosphate buffer, 500 mM sodium chloride, pH 7.0 [0381] Column:
Dionex ProPac WCX-10, 4.times.250 mm [0382] Column Temperature:
Ambient [0383] Flow Rate: 1.0 mL/min [0384] Wavelength: Signal, 214
nm; Reference, 360 nm [0385] Injection volume: 5 .mu.L [0386]
Sample Preparation: No dilution [0387] Percent purity reported
based on integrated area percent of main peaks identified for both
Antagonist A and ranibizumab.
[0388] (3) AEX-HPLC (Analysis of Antagonist A) [0389] Mobile Phase
A: 10 mM phosphate buffer, pH 7.0 [0390] Mobile Phase B: 10 mM
phosphate buffer, 500 mM sodium chloride, pH 7.0 [0391] Column:
Dionex DNA Pac PA-100, 4.times.250 mm [0392] Column Temperature:
40.degree. C. [0393] Flow Rate: 1.2 mL/min [0394] Wavelength:
Signal, 258 nm; Reference. 360 nm [0395] Injection volume: 5 .mu.L
[0396] Sample Preparation: No dilution [0397] Percent purity
reported based on integrated area percent of main peaks identified
for both Antagonist A and ranibizumab.
[0398] (4) nH [0399] VWR symphony SB70P
[0400] (5) Visual Observation [0401] Photos taken from Sony
Cyber-shot DSC-H9 Digital Still Camera (8.1 Mega pixels)
[0402] (6) Osmolarity [0403] Advanced Instruments, Inc. The
Advanced Osmometer Model 3D3
Stability Overview
[0404] The effects of both agitation (4 hours) and the various
storage temperatures (4.degree. C., 25.degree. C., and 37.degree.
C.) on various Antagonist A and ranibizumab compositions were
analyzed. Throughout the study, all of the compositions tested were
able to maintain their target pH values, i.e., titrated initial pH,
through all storage and stress conditions.
[0405] Stability Indicating Assays
[0406] Composition F2 developed visible precipitation during
storage at 37.degree. C. after two weeks (data not shown). No other
assays were performed for quantitative measurement of the
precipitation.
[0407] The degradation of Antagonist A during storage was
effectively analyzed by AEX-HPLC (FIG. 1). The formation of
pre-peaks and post-peaks was observed when samples were incubated
at elevated temperature (FIG. 1). In composition F2, the AEX-HPLC
purity of Antagonist A decreased by nearly 20% during storage for 8
weeks at 37.degree. C.
[0408] WCX-HPLC was also effective at characterizing the
degradation of ranibizumab during storage (FIG. 2). The formation
of both pre-peaks and post-peaks was observed when ranibizumab was
incubated at elevated temperature (FIG. 2).
[0409] The SE-HPLC assay was useful for characterizing soluble
aggregation or fragmentation of ranibizumab (FIG. 3). Antagonist A
did not show significant changes by SE-HPLC, although the
resolution of its aggregated form may be beyond the capacity of
Tosoh TSKgel G3000SWXL column. Ranibizumab underwent aggregation or
fragmentation in composition F3 and composition F5 during storage
at 37.degree. C. (FIG. 3).
[0410] Effect of Agitation on Stability
[0411] All compositions listed in Table 1 underwent 4 hours of
agitation, with a set of non-agitated control compositions left at
room temperature. No differences were seen between control and
agitated samples on any analytical methods (data not shown).
[0412] Effect of Storage Temperature on Stability
[0413] Storage at 37.degree. C. produced significant albeit varying
levels of degradation of both Antagonist A and ranibizumab in the
various compositions investigated. By two weeks, composition F2
developed precipitation (data not shown). All other compositions
remained clear through eight weeks, and up to 12 weeks for several
compositions (Compositions F1, F4, F6, F8, and F11).
[0414] After two weeks at 37.degree. C., Antagonist A purity in
composition F2 had decreased by nearly 20% based on AEX-HPLC.
Compositions F3 and F5 also revealed increased Antagonist A
degradation after four weeks under the same storage conditions
(FIG. 4). By eight weeks, it appeared that composition F8 offered
greater protection to Antagonist A than did F6 and F7. By 12 weeks,
F8 continued to display higher purity of Antagonist A (FIG. 4).
[0415] Composition F2 also could not prevent degradation of
ranibizumab, as WCX-HPLC detected nearly 20% loss of purity by 2
weeks (FIG. 5). By the fourth week, many compositions (F3, F5, F7,
F8, F9, and F10) exhibited significant degradation of ranibizumab
in comparison to F6 (FIG. 5). Composition F6 maintained the best
purity of ranibizumab for up to 12 weeks (FIG. 5).
[0416] Based on the results from 2, 8, and 12 weeks at 4.degree.
C., which showed a single peak for the native form of ranibizumab,
all compositions displayed similar purity profiles of Antagonist A
and ranibizumab for up to 4 weeks by SE-HPLC (FIG. 14 and FIG. 15).
No significant change was observed for Antagonist A at all storage
conditions, including the 12 week storage at 37.degree. C. (FIG.
6). However, ranibizumab underwent aggregation during storage at
25.degree. C. and 37.degree. C. No significant aggregation was
observed with a diluted form of commercial Lucentis.RTM. (F4) under
the same storage condition (FIG. 7).
[0417] All compositions showed better visual stability during
storage at 25.degree. C. than at 37.degree. C. Over 8 weeks, all
compositions remained clear. Two compositions (F6 and F8) remained
clear at the additional 12 week time point.
[0418] For the first four weeks at 25.degree. C., all compositions
maintained comparable Antagonist A purity as characterized by
AEX-HPLC (FIG. 8). Composition F2 underwent a significant increase
in Antagonist A degradation by 8 weeks (FIG. 8). Also, compositions
F3 and F5 displayed considerable decreases in purity over the same
timeframe (FIG. 8). Compositions F6, F7, and F8 were able to
maintain the purity of Antagonist A for up to 12 weeks (FIG.
8).
[0419] WCX-HPLC analysis of ranibizumab displayed subtle yet
distinctive changes in purity profiles between the compositions.
After two weeks of storage at 25.degree. C., composition F2
developed considerable degradation of ranibizumab (FIG. 9). The
remaining compositions maintained comparable purity of ranibizumab
until eight weeks, when the pH 8.0 compositions (F9 and F10)
revealed a considerable decrease in purity of ranibizumab (FIG. 9).
Composition F6 was able to prevent degradation of ranibizumab at
25.degree. C. as determined by WCX-HPLC analysis (FIG. 9).
[0420] Other than its inherent variability, the SE-HPLC assay
showed no significant change of Antagonist A profile during storage
at 25.degree. C. (FIG. 10). In general, all compositions appeared
to prevent aggregation or fragmentation of Antagonist A over eight
weeks, and over twelve weeks for compositions F6 and F8 (FIG. 10).
Compositions F8 and F6 maintained good ranibizumab purity for
twelve weeks at 25.degree. C. (FIG. 11).
[0421] Antagonist A and ranibizumab remained stable in most
compositions at 4.degree. C. All compositions remained clear by
visual inspection. Furthermore, most compositions maintained
comparable purity to the starting material by all HPLC methods
(FIG. 12-15), except for F2, F3, and F5, which yielded substantial
amounts of soluble aggregates of ranibizumab (FIG. 15).
Effect of Composition Characteristics/Components on Stability
[0422] To determine the effect that pH and different composition
components have on the stability of Antagonist A and ranibizumab,
Antagonist A and ranibizumab were coformulated at various pH levels
(5.0-8.0) and with different tonicity modifiers (sodium chloride
and sorbitol). This section describes the effects of pH and
composition components on the stability of one or both of
Antagonist A and ranibizumab when stored at various
temperatures.
[0423] Effect of pH on Stability
[0424] The effect of pH on the stability of Antagonist A and
ranibizumab was best differentiated by storage at 37.degree. C. in
both sorbitol-containing and NaCl containing compositions (FIG.
16). Based on AEX-HPLC, degradation of Antagonist A was inversely
correlated with pH, with the greatest degradation at pH 5.0 (FIG.
16). Changes in pH caused less significant changes to the purity
profile of ranibizumab in sorbitol-containing compositions. Based
on WCX-HPLC, formulating at pH 5.0 yielded faster degradation of
ranibizumab after four weeks at 37.degree. C., but yielded similar
degradation to the pH 6.0 compositions after eight weeks at
37.degree. C. (FIG. 17). Ranibizumab was least degraded at pH 6.0
among the NaCl-containing compositions, while pH 7.0 was the best
among the sorbitol-containing compositions. Using SE-HPLC for
evaluation of both Antagonist A (FIG. 18) and ranibizumab (FIG.
19), the aggregation rate of ranibizumab was slowest in
compositions at pH 7.0, while no changes were observed for
Antagonist A degradation.
[0425] Effect of Tonicity Modifier on Stability
[0426] The effect of tonicity modifiers on the stability of
Antagonist A and ranibizumab was differentiated by comparing the
results from 37.degree. C. storage. As characterized by AEX-HPLC,
Antagonist A remained more stable in NaCl compositions than in
sorbitol compositions at pH 5.0-7.0 over 8 weeks (FIG. 20). At pH
8.0, no discernable difference could be made between compositions
containing sodium chloride or sorbitol over 4 weeks (FIG. 21). For
ranibizumab compositions, as characterized by WCX-HPLC, sodium
chloride compositions outperformed sorbitol compositions across the
pH range tested (pH 5.0-8.0) (FIG. 22). The superior performance of
sodium chloride compositions in stabilizing ranibizumab was also
revealed by SE-HPLC (FIG. 23). For compositions with both tonicity
modifiers, the level of soluble aggregation was lowest at pH 7.0
and highest at pH 5.0 (FIG. 23).
[0427] Stability of 1:1 Mixture of Antagonist A and
Lucentis.RTM.
[0428] Another aspect of the study involved characterizing the
effect of admixing Antagonist A and commercial Lucentis.RTM.. To
accomplish this. Antagonist A was diluted to 6 mg/mL from its
original concentration of 30 mg/mL in a composition of 10 mM sodium
phosphate and 150 mM NaCl, pH 7.3, followed by combining the
resulting composition with an equal volume (1:1) of commercial
Lucentis.RTM..RTM. (10 mg/mL). Stability of the 1:1 mixture (F1)
was examined by storage at 37.degree. C., and was compared to F1
and F4 alone at similar concentrations and storage
temperatures.
[0429] For Antagonist A, SE-HPLC analysis indicated that the
stability of Antagonist A in the 1:1 mixture. F11, is comparable to
F1 alone over twelve weeks at 37.degree. C. (FIG. 24). Although it
appeared by AEX-HPLC that Antagonist A underwent faster degradation
in the 1:1 mixture at earlier time points during storage at
37.degree. C., both F1 and the 1:1 mixture (F11) displayed
comparable purity by 12 weeks (FIG. 25). No difference in the
AEX-HPLC purity profile was observed when the samples were stored
at 25.degree. C. (FIG. 25).
[0430] Ranibizumab encountered more stability issues in the 1:1
mixture than Antagonist A did at similar storage conditions.
Ranibizumab in F11 maintained a comparable WCX-HPLC profile to
ranibizumab in F4 up to 4 weeks storage at 37.degree. C., after
which ranibizumab underwent faster degradation in the mixture (F11)
(FIG. 26). Ranibizumab, however, remained fairly stable in the
mixture when the samples were stored at 25.degree. C. (FIG. 26). No
significant difference in the WCX-HPLC purity profile of
ranibizumab was observed between the mixture and F4 at 25.degree.
C. SE-HPLC revealed a noticeable increase in aggregated ranibizumab
in the 1:1 mixture after 8 weeks of storage at 37.degree. C.
compared to F4 (FIG. 27a). The aggregation in the mixture was
substantially lower when stored at 25.degree. C., and was not
observed at 4.degree. C. (FIG. 27b-c).
[0431] Stability of Composition F6
[0432] AEX-HPLC analysis of Antagonist A indicated that the F6
composition maintained the purity of Antagonist A up to twelve
weeks at 25.degree. C., and up to at least sixteen weeks (the
latest timepoint tested) at 4.degree. C. (FIG. 28). At 37.degree.
C. Antagonist A degradation was observed as early as two weeks
(FIG. 28). Formulating with F6 helped protect ranibizumab from
degradation at 37.degree. C. for up to four weeks before a
significant decrease in purity by WCX-HPLC developed by eight weeks
(FIG. 29). However, ranibizumab was stable in composition F6 for up
to at least twelve weeks at 25.degree. C. and for up to at least
sixteen weeks at 4.degree. C., without any substantial loss in
purity by WCX-HPLC (FIG. 29).
[0433] SE-HPLC results indicated that Antagonist A remains stable
over sixteen weeks at all storage conditions (FIG. 30). For
ranibizumab, no significant aggregation was observed when incubated
for twelve weeks at 37.degree. C. in the F6 composition (FIG. 31).
The F6 composition performed better when stored at either 4.degree.
C. or 25.degree. C., with comparable purity over eight weeks at
both temperatures. Aggregation of ranibizumab at 25.degree. C. and
37'C was faster in the F6 composition than in F4.
[0434] From these ranging studies, composition F6, on average,
demonstrated the best stability over all storage temperatures and
analysis methods employed for this study.
Example 2
Stability of Compositions Comprising Antagonist A and
Bevacizumab
[0435] The stability of Antagonist A in a composition that also
includes the anti-VEGF monoclonal antibody (mAb) bevacizumab was
examined under a range of conditions. Various pHs (4.0-8.0) and
tonicity modifiers (sodium chloride, sorbitol, and trehalose) were
used to optimize the composition stability of Antagonist A and
bevacizumab when stored at various temperatures (4.degree. C.,
25.degree. C., and 37.degree. C.) and against a physical stress
(agitation). The stability of Antagonist A and bevacizumab was
characterized by visual observation, pH measurement, and various
HPLC methods (anion exchange [AEX-HPLC], weak cation exchange
[WCX-HPLC], and size exclusion [SE-HPLC]).
[0436] Antagonist A was compatible with bevacizumab with no
discernable stability issue when both were combined together in
certain of the compositions tested. Based on the results from a 24
week stability study, the best stability was observed with
Composition F19. In the F19 composition, both Antagonist A and
bevacizumab remained stable throughout 24 weeks at 4.degree. C.,
and for up to at least 4 weeks at 25.degree. C.
Composition Parameters
[0437] The following composition parameters were examined: [0438]
(1) pH: 4.0, 5.0, 6.0, 6.2, 6.3, 7.0, 7.3, 8.0 [0439] (2) Buffers:
Acetate, Phosphate, and Tris [0440] (3) Tonicity Modifiers: Sodium
Chloride, Sorbitol, and Trehalose [0441] (4) Surfactants:
Polysorbate 20 [0442] (5) Antagonist A Concentration: 30 mg/mL, 15
mg/mL, and 3 mg/mL
[0443] The following parameters were fixed: [0444] (1) Fill volume
was 300 IL in modified 3 cc vials provided by Ophthotech Corp.
(obtained from Mglas AG, Munnerstadt, Germany) [0445] (2) The
concentration of bevacizumab was 12.5 mg/mL.
[0446] Table 3 below summarizes the composition matrix used in this
study.
TABLE-US-00007 TABLE 3 Composition Matrix for Antagonist A:
Bevacizumab Compositions Antagonist A Bevacizumab Concentration
Concentration Comp. Buffer pH Tonicity Modifier (mg/mL, oligo wt.)
(mg/mL) Surfactant F12 10 mM 7.3 150 mM Sodium 30 0.0 0% Phosphate
Chloride F13 50 mM 4 5% (w/v) Sorbitol 3 12.5 0.02% Acetate
Polysorbate 20 F14 50 mM 4 130 mM Sodium 3 12.5 0.02% Acetate
Chloride Polysorbate 20 F15 50 mM 5 5% (w/v) Sorbitol 3 12.5 0.02%
Acetate Polysorbate 20 F16 50 mM 5 130 mM Sodium 3 12.5 0.02%
Acetate Chloride Polysorbate 20 F17 50 mM 6 5% (w/v) Sorbitol 3
12.5 0.02% Phosphate Polysorbate 20 F18 50 mM 6.2 6% (w/v) 0 12.5
0.02% Phosphate Trehalose Polysorbate 20 F19 50 mM 6 130 mM Sodium
3 12.5 0.02% Phosphate Chloride Polysorbate 20 F20 50 mM 7 5% (w/v)
Sorbitol 3 12.5 0.02% Phosphate Polysorbate 20 F21 50 mM 7 130 mM
Sodium 3 12.5 0.02% Phosphate Chloride Polysorbate 20 F22 50 mM 8
5% (w/v) Sorbitol 3 12.5 0.02% Tris Polysorbate 20 F23 50 mM 8 130
mM Sodium 3 12.5 0.02% Tris Chloride Polysorbate 20 F24 30 mM 6.3
75 mM sodium 15 12.5 0.02% Phosphate Chloride + 3% Polysorbate
(w/v) Trehalose 20 F25 10 mM 7.3 150 mM Sodium 3 0.0 0% Phosphate
Chloride F26 30 mM 6.3 75 mM sodium 3 12.5 0.02% Phosphate Chloride
+ 3% Polysorbate (w/v) Trehalose 20
Sample Preparation
[0447] An Antagonist A stock solution was prepared at 6 mg/mL in 10
mM phosphate, 150 mM NaCl, and pH 7.3. The resulting stock solution
was admixed 1:1 with commercial Avastin.RTM. (25 mg/mL), resulting
in final concentrations of 3 mg/mL Antagonist A and 12.5 mg/mL
bevacizumab (Composition F26). The composition was placed in 10 kDa
molecular weight cutoff dialysis cassettes and dialyzed
.about.1,000,000-fold against the various composition buffers
listed in Table 3 (Comp. Nos. F13-F17, F19-F23). Exceptions include
the following: [0448] Composition F12 needed no additional dilution
or dialysis. [0449] Commercial Avastin.RTM. was diluted 1:1 with 50
mM phosphate buffer (pH 6.2) containing 6% (w/v) trehalose and
0.02% (w/v) polysorbate 20 to provide Composition F18. [0450]
Composition F24 was made by admixing 1:1 of composition F12 with
commercial Avastin.RTM.. [0451] Composition F25 was created with
10.times. dilution of Composition F12 with 10 mM phosphate buffer
(pH 7.3) containing 150 mM NaCl.
Stress Studies
[0452] The compositions of Table 3 were tested under the following
stress conditions:
TABLE-US-00008 TABLE 4 Stress Conditions Stress Conditions Time
points Storage 4.degree. C. 0, 2, 4, 8, 12, and 24 weeks 25.degree.
C. 2, 4, 8, 12, and 24 weeks 37.degree. C. 2, 4, 8, and 12 weeks
Agitation 4 hours
Analytical Methods
[0453] In order to analyze degradation products generated under
stress, the following stability-indicating assays were developed
and used in this study.
[0454] (1) SE-HPLC (Analysis of Antagonist A and bevacizumab)
[0455] Mobile Phase: 50 mM phosphate buffer, 100 mM sodium
chloride, pH 7.0 [0456] Column: TOSOH TSKgel G3000SW.sub.XL [0457]
Column Temperature: Ambient [0458] Flow Rate: 1.0 mL/min [0459]
Wavelength: Signal, 214 nm; Reference, 360 nm [0460] Injection
volume: 1 .mu.L [0461] Sample Preparation: [0462] 10.times.
dilution in Milli-Q water for 30 mg/mL aptamer samples [0463] No
dilution for other samples [0464] Percent purity reported based on
integrated area percent of main peaks identified for both
Antagonist A and bevacizumab
[0465] (2) WCX-HPLC (Analysis of bevacizumab) [0466] Mobile Phase
A: 10 mM phosphate buffer, pH 7.0 [0467] Mobile Phase B: 10 mM
phosphate buffer, 500 mM sodium chloride, pH 7.0 [0468] Column:
Dionex ProPac WCX-10, 4.times.250 mm [0469] Column Temperature:
Ambient [0470] Flow Rate: 1.0 mL/min [0471] Wavelength: Signal, 214
nm; Reference, 360 nm [0472] Injection volume: 10 .mu.L [0473]
Sample Preparation: 10.times. dilution in Milli-Q water [0474]
Percent purity reported based on integrated area percent of the
main peak identified for bevacizumab
[0475] (3) AEX-HPLC (Analysis of Antagonist A) [0476] Mobile Phase
A: 10 mM phosphate buffer, pH 7.0 [0477] Mobile Phase B: 10 mM
phosphate buffer, 500 mM sodium chloride, pH 7.0 [0478] Column:
Dionex DNA Pac PA-100, 4.times.250 mm [0479] Column Temperature:
40.degree. C. [0480] Flow Rate: 1.2 mL/min [0481] Wavelength:
Signal, 258 nm; Reference, 360 nm [0482] Injection volume: 5 .mu.L
[0483] Sample Preparation: [0484] 10.times. dilution in Milli-Q
water for 3.0 mg/mL aptamer samples [0485] 50.times. dilution in
Milli-Q water for 15 mg/mL aptamer samples [0486] 100.times.
dilution in Milli-Q water for 30 mg/mL aptamer samples [0487]
Percent purity reported based on integrated area percent of the
main peak identified for Antagonist A
[0488] (4) pH [0489] VWR symphony SB70P
[0490] (5) Visual Observation [0491] Photos taken from Sony
Cyber-shot DSC-H9 Digital Still Camera (8.1 Mega pixels)
[0492] (6) Osmolarity (at time point zero) [0493] Advanced
Instruments, Inc. Advanced Osmometer Model 3D3
Stability Overview
[0494] This section describes the effect of both agitation (4
hours) and the storage at various temperatures (4.degree. C.,
25.degree. C., and 37.degree. C.) on Antagonist A and bevacizumab.
Throughout the study, each composition was able to maintain
targeted pH values through all physical stresses.
[0495] Stability Indicating Assays
[0496] By visual observation, it was noted that compositions F15,
F16, and F24 developed precipitation during 2 weeks of storage at
37.degree. C. (data not shown). Due to the limited volumes
available for the study, no other assay was performed for
quantitative measurement of precipitation.
[0497] The stability of Antagonist A during storage was effectively
analyzed by AEX-HPLC. The formation of both pre-peaks and post
peaks was observed when Antagonist A in certain compositions was
incubated at elevated temperature of 37.degree. C. For example, in
Composition F14, the AEX-HPLC purity of Antagonist A had decreased
by nearly 50% during storage for 2 weeks at 37.degree. C. (FIG.
32).
[0498] WCX-HPLC was also effective in characterizing the stability
of bevacizumab. The formation of both pre-peaks and post peaks was
observed when bevacizumab in certain compositions was incubated at
a temperature of 25.degree. C. For example, in Composition F22,
bevacizumab purity decreased nearly 30% during 8 weeks of storage
at 25.degree. C. (FIG. 33).
[0499] SE-HPLC proved useful for characterizing soluble aggregation
or fragmentation of bevacizumab. Antagonist A did not show
significant changes by SE-HPLC, although the resolution of its
aggregated form may be beyond the capacity of TSKgel G3000SWXL
column due to assay sensitivity to stability. Degradation of
bevacizumab was seen in composition F15 after 8 weeks of storage at
37.degree. C. (FIG. 34).
[0500] Effect of Agitation on Stability
[0501] The effect of agitation on one or both of Antagonist A and
bevacizumab was assesed. The compositions listed in Table 3 were
agitated for 4 hours with an in-house agitator, while a control set
of compositions was left unagitated at room temperature. No
differences in visual observation, pH, AEX-HPLC, and WCX-HPLC were
observed between agitated samples and controls (data not shown).
However, SE-HPLC, which assesses aggregation or fragmentation of
both Antagonist A and bevacizumab, displayed slight variations
between agitated and control samples in F23 and F24 samples (Table
5 and Table 6). After 4 hours of agitation, more soluble aggregates
(pre-Antagonist A peak and pre-bevacizumab peak) formed in the F23
samples and the direct 1:1 mixture of 30 mg/mL Antagonist A and 25
mg/mL Avastin.RTM. (F24). This suggests that formulating at pH 8.0
with sodium chloride, or having a higher concentration of
Antagonist A coformulated with bevacizumab, leads to Antagonist A
or bevacizumab forming soluble aggregates or fragments during shear
stress. The other compositions appeared to maintain the integrity
of Antagonist A and bevacizumab as determined by SE-HPLC. These
results suggest that except under the conditions noted above, no
apparent degradation of coformulated Antagonist A or bevacizumab
was induced by agitation.
TABLE-US-00009 TABLE 5 SE-HPLC results for samples before agitation
bevacizumab Antagonist A Area (%) Area (%) Pre- Post- Conc. (mg/mL)
Pre- Ant. Ant. bev. bev. bev. Comp. Ant. A bev. A Peak A Peak Pk
Peak Peak F12 30.0 0.0 0.3 99.7 NA NA NA F13 3.0 12.5 4.6 95.4 4.6
89.3 6.1 F14 3.0 12.5 6.2 93.8 4.0 89.6 6.4 F15 3.0 12.5 5.6 94.4
3.9 90.1 6.0 F16 3.0 12.5 4.2 95.8 4.5 89.4 6.1 F17 3.0 12.5 3.6
96.4 4.0 90.7 5.3 F18 0.0 12.5 NA NA 4.7 90.1 5.3 F19 3.0 12.5 2.5
97.5 4.4 91.1 4.5 F20 3.0 12.5 2.4 97.6 9.5 86.1 4.4 F21 3.0 12.5
2.6 97.4 13.5 82.0 4.5 F22 3.0 12.5 3.0 97.0 13.8 81.6 4.6 F23 3.0
12.5 3.1 96.9 16.7 78.0 5.2 F24 15.0 12.5 2.3 97.7 16.8 76.1 7.0
F25 3.0 0.0 0.7 99.3 NA NA NA F26 3.0 12.5 1.7 98.3 7.2 88.0 4.8
"Ant. A" is Antagonist A; "bev." is bevacizumab; "NA" means not
applicable
TABLE-US-00010 TABLE 6 SE-HPLC results for samples after 4 hours
agitation bevacizumab Antagonist A Area (%) Area (%) Pre- Post-
Conc. (mg/mL) Pre- Ant. Ant. bev. bev. bev. Comp. Ant. A bev. A
Peak A Peak Peak Peak Peak F12 30.0 0.0 0.3 99.7 NA NA NA F13 3.0
12.5 5.4 94.6 4.0 90.0 6.0 F14 3.0 12.5 6.0 94.0 3.9 90.1 6.0 F15
3.0 12.5 3.4 96.6 3.2 91.6 5.1 F16 3.0 12.5 3.4 96.6 4.0 89.9 6.2
F17 3.0 12.5 2.4 97.6 4.2 90.5 5.4 F18 0.0 12.5 NA NA 4.1 90.8 5.2
F19 3.0 12.5 1.7 98.3 3.9 91.7 4.4 F20 3.0 12.5 2.7 97.3 9.2 86.2
4.7 F21 3.0 12.5 4.4 95.6 13.3 82.2 4.5 F22 3.0 12.5 4.8 95.2 13.4
82.0 4.6 F23 3.0 12.5 7.1 92.9 16.0 78.7 5.3 F24 15.0 12.5 5.4 94.6
20.7 74.6 4.7 F25 3.0 0.0 1.1 98.9 NA NA NA F26 3.0 12.5 2.3 97.7
7.3 88.4 4.3 "Ant. A" is Antagonist A; "bev." is bevacizumab; "NA"
means not applicable
[0502] Effect of Storage Temperature on Stability
[0503] During the 24-week study, the compositions listed in Table 3
were placed in 4.degree. C., 25.degree. C., and 37.degree. C.
stability chambers to study the effects of temperature on one or
both of Antagonist A and bevacizumab stability. Both Antagonist A
and bevacizumab exhibited greater degradation with increasing
storage temperature, based on the chromatographic assays.
[0504] Storage at 37.degree. C. induced significantly elevated
levels of degradation of Antagonist A and bevacizumab. By 2 weeks,
precipitation of Antagonist A or bevacizumab was seen in F15, F16,
and F24 (data not shown). By 4 weeks. F14 also began showing
insoluble aggregation of Antagonist A or bevacizumab (data not
shown). All other composition remained clear throughout 12
weeks.
[0505] AEX-HPLC revealed significant Antagonist A degradation in
composition samples at pH 4.0 and 5.0 (F13, F14, F15, and F16),
while coformulated samples in F17 displayed better stability (FIG.
35). Antagonist A maintained comparable purity at pH 6.0-7.0
through 12 weeks of storage at 37.degree. C., with the exception of
F20 and F26, where decreases in Antagonist A purity were observed
at 12 weeks (FIG. 36).
[0506] After 2 weeks of storage at 37.degree. C., WCX-HPLC revealed
significant decreases in bevacizumab purity in pH 4.0 composition
(F13 and F14), displaying low to no intact bevacizumab remaining
(FIG. 37). Accelerated degradation was observed through 12 weeks of
storage at 37.degree. C. in all other composition except for F19,
which consistently revealed slower degradation than other
composition (FIG. 38).
[0507] SE-HPLC revealed the formation of soluble aggregates in the
stressed samples. For Antagonist A, 2 weeks of storage at
37.degree. C. caused composition at pH 4.0-5.0 to rapidly form
soluble aggregates (FIG. 39). Antagonist A formulated in F17 also
showed soluble aggregation but at a lower rate (FIG. 39). By the
fourth week, most of the Antagonist A compositions displayed lower
Antagonist A purity, with the exception of F19 and the two 1:1
mixtures (F24 and F26), which were able to maintain high Antagonist
A purity (FIG. 40). This trend was maintained until Week 12, when
F26 revealed slightly reduced Antagonist A purity, leaving F19 as
the composition of choice for Antagonist A with respect to
stability. For bevacizumab, formulating outside of pH 6.0 caused a
significant decrease in the mAb purity (FIG. 41). This trend
continued throughout 12 weeks of storage at 37.degree. C., leaving
F19 as the composition providing bevacizumab with the greatest
stability (FIG. 42).
[0508] Some compositions provided better stability during storage
at 25.degree. C. All of the compositions remained clear after 24
weeks at 25.degree. C. except for F14, in which precipitation was
observed at 8 weeks (data not shown).
[0509] Based on AEX-HPLC, formulating Antagonist A at pH 4.0 (F13
and F14) caused degradation of the aptamer after just 2 weeks of
storage (FIG. 43). F15 revealed degradation at 4 weeks of
25.degree. C. storage; however, F16 exhibited improved stability up
to 8 weeks (FIG. 43). Formulating Antagonist A at pH 6.0-8.0
maintained comparable stability through 8 weeks of storage at
25.degree. C., and up to at least 24 weeks storage at 4.degree. C.
with compositions F19, F20, F21, and F23 (FIG. 44).
[0510] WCX-HPLC indicated that pH had the opposite effect on the
stability of bevacizumab compared to Antagonist A. After 2 weeks at
25.degree. C., pH 8.0 samples revealed substantial degradation of
bevacizumab (FIG. 45 and FIG. 46). By 4 weeks at 25.degree. C., pH
4.0 and pH 7.0 compositions began displaying signs of bevacizumab
degradation (FIG. 45 and FIG. 46). Compositions at pH 5.0-6.0
provided comparable stability of bevacizumab up to 12 weeks at
25.degree. C., at which time all leading candidates displayed
accelerated signs of degradation. However, the F19 composition, at
pH 6.0, did not undergo further accelerated degradation of
bevacizumab from 12 to 24 weeks of storage at 25.degree. C. (FIG.
45 and FIG. 46).
[0511] Similar degradation trends seen in AEX-HPLC and WCX-HPLC
were observed by SE-HPLC. Antagonist A formulated at pH 4.0 was
unable to maintain Antagonist A purity when stored at 25.degree. C.
(FIG. 47). By 8 weeks, Antagonist A formulated at pH 5.0 underwent
significant aggregation or fragmentation (FIG. 47). Formulating
Antagonist A in the pH range of 6.0-8.0 provided for comparable
purity through up to at least 24 weeks of 25.degree. C. storage
(FIG. 21). The purity of bevacizumab depended on the pH of the
composition and the concentration of Antagonist A in the
composition. After 4 weeks of storage at 25.degree. C., formulating
at pH 4.0 and pH 8.0 caused an accelerated decrease in the purity
of bevacizumab (FIG. 49 and FIG. 50). Under the same time and
storage conditions, Antagonist A coformulated at 15 mg/mL appeared
to adversely affect the purity of bevacizumab (FIG. 49).
Compositions at pH 5.0-7.0 provided for better stability at
25.degree. C. over 8 weeks (FIG. 49 and FIG. 50). Further time
points revealed that leading compositions (pH 6.0 and 7.0) were
able to maintain comparable purity (FIG. 50).
[0512] Storage at 4.degree. C. provided the best stability for most
compositions during this study. Visual observation revealed no
insoluble aggregation during 4.degree. C. storage for up to at
least 24 weeks for compositions F19, F20, F21, and F23.
[0513] For Antagonist A, all compositions maintained comparable
purity by AEX-HPLC after eight weeks of storage, and through 24
weeks at 4.degree. C. with compositions F19, F20, F21, and F23
(FIG. 51). However, as observed by WCX-HPLC, formulating
bevacizumab at pH 8.0 caused a considerable increase in degradation
after eight weeks at 4.degree. C., a trend which carried on through
24 weeks (FIG. 52).
[0514] SE-HPLC revealed some fragmentation of Antagonist A or
aggregation of bevacizumab in a few compositions. For Antagonist A,
most compositions maintained their purity up to 8 weeks at
4.degree. C., while compositions at pH 4.0-5.0 revealed significant
losses of purity (FIG. 53). Compositions F19. F20, F21 and F23
maintained comparable Antagonist A purity up to 12 weeks of
4.degree. C. storage; however, after 12 and 24 weeks, Antagonist A
purity in F23 decreased substantially, while that of the other
three selected compositions remained similarly elevated (FIG. 54).
Formulating at pH 8.0 caused formation of soluble aggregates of
bevacizumab during initial dialysis; however, storage at 4.degree.
C. maintained the purity of bevacizumab through at least eight
weeks, similar to the other compositions (FIG. 55). The one
exception was composition F24, where the concentration of
Antagonist A at 15 mg/mL affected the purity of bevacizumab over
the eight weeks of storage (FIG. 55).
Effect of Composition Characteristics/Components on Stability
[0515] Antagonist A and bevacizumab were coformulated at varying pH
and with different tonicity modifiers in order to determine the
effects of these factors on stability. This section describes the
effects of the composition on the stability of one or both of
Antagonist A and bevacizumab.
[0516] Effect of pH on Stability
[0517] The effects of pH on stability of Antagonist A and
bevacizumab were differentiated by storage at 37.degree. C. As
observed by AEX-HPLC, Antagonist A was stable at 37.degree. C. in
the pH 7.0 and pH 8.0 sorbitol-containing compositions F20 and F22
in contrast to the pH 4.0-6.0 sorbitol-containing compositions F13,
F15, and F17, where accelerated degradation occurred (FIG. 56). For
bevacizumab, as observed by WCX-HPLC, sorbitol-containing
compositions outside of pH 5.0-6.0 (F13, F20, and F22 exhibited
accelerated degradation of bevacizumab at 37.degree. C. (FIG. 57).
Similar to the AEX-HPLC results, SE-HPLC revealed that Antagonist A
in sorbitol-containing compositions F13 and F15 (pH 4.0-5.0)
underwent fragmentation or aggregation at 37.degree. C. (FIG. 58).
However, despite the degradation seen by WCX-HPLC for
sorbitol-containing compositions outside the range of pH 5.0-6.0,
SE-HPLC revealed that bevacizumab underwent slower aggregation or
fragmentation in sorbitol-containing compositions at pH 5.0-8.0
when stored at 37.degree. C. (FIG. 59). SE-HPLC of the pH 4.0
sorbitol-containing composition F13 stored at 37.degree. C.
revealed substantial degradation of bevacizumab. Formulating at pH
6.0 (F17) appeared to maintain the purity of bevacizumab better
than the other pH levels assayed for sorbitol-containing
compositions (FIG. 58 and FIG. 59).
[0518] Effect of Tonicity Modifier on Stability
[0519] The effect of tonicity modifiers on the stability of
Antagonist A and bevacizumab was differentiated by storage at
37.degree. C. The benefits of either sorbitol or sodium chloride
depended on pH of the composition.
[0520] At pH 5.0 and 6.0, Antagonist A underwent degradation in
sorbitol compositions (F15 and F17) throughout the eight week study
as observed by AEX-HPLC (FIG. 60). However, compositions at these
pH levels with sodium chloride as tonicity modifier (F16 and F19)
did not undergo such degradation (FIG. 60). The pH 4.0 composition
containing sodium chloride (F14) proved to have reduced stability
after 4 weeks of accelerated stress, resulting in sorbitol being
the superior tonicity modifier at pH 4.0 (FIG. 60). At pH 7.0 and
pH 8.0, compositions with either sodium chloride or sorbitol as
tonicity modifier (F20, F21, F22, and F23) maintained comparable
stability. Analysis of bevacizumab by WCX-HPLC revealed that
formulating with sodium chloride from pH 6.0-7.0 improved stability
relative to sorbitol (FIG. 61). However, the opposite was true for
pH 5.0 compositions, where sorbitol limited degradation relative to
sodium chloride for 4 weeks storage at 37.degree. C. (FIG. 61). By
SE-HPLC, Antagonist A stability was impacted by the presence of
sodium chloride or sorbitol, while the stability of bevacizumab
remained comparable between both tonicity modifiers. For pH 5.0-6.0
compositions, the presence of sodium chloride protected Antagonist
A from aggregation or fragmentation better than sorbitol (FIG. 62).
With the other pHs assayed, Antagonist A displayed lower purity at
pH 4.0 with sorbitol (FIG. 62). Antagonist A formulated at pH 7.0
and pH 8.0 (FIG. 62) and bevacizumab formulated at pH 4.0, pH 7.0,
and pH 8.0 (FIG. 63) maintained comparable purity with either
sorbitol or sodium chloride as tonicity modifier.
[0521] Effect of 1:1 Mixture on Stability
[0522] Another parameter analyzed was the effect of mixing
Antagonist A and commercial bevacizumab. Also, compositions
containing different concentrations of Antagonist A with a fixed
concentration of bevacizumab (1:1 Mix (F24) and 1:1 Mix (F26) were
analyzed. Also, stressing the compositions at 37.degree. C.
provided information on the degradation of both Antagonist A and
bevacizumab.
[0523] For Antagonist A alone, formulating at 30 mg/mL (F12) or 3
mg/mL (F25) produced no difference in stability profiles by
AEX-HPLC and SE-HPLC. Upon mixing commercial Avastin.RTM. with
varying Antagonist A concentrations (15 mg/mL and 3 mg/mL).
Antagonist A in both compositions maintained comparable stability
up to 8 weeks at 37.degree. C., whereas formulating Antagonist A at
15 mg/mL with 12.5 mg/mL of bevacizumab produced slight degradation
as observed by AEX-HPLC (FIG. 64).
[0524] Even though the concentration of bevacizumab was constant in
all of the compositions in this study, the varying concentrations
of Antagonist A affected the stability of bevacizumab. After 8
weeks storage at 37.degree. C., WCX-HPLC revealed minor differences
in the degradation profile of bevacizumab when formulated with
either 3 mg/mL Antagonist A (F26) or 15 mg/mL Antagonist A (F24)
(FIG. 65). By SE-HPLC, no significant differences in purity
profiles were seen between Antagonist A at 30 mg/mL and 3 mg/mL
compared to direct 1:1 mixing at two concentrations (F24 and F26)
(FIG. 66). However, for bevacizumab, compositions with 15 mg/mL
Antagonist A (F24) produced more soluble aggregation and
fragmentation of the bevacizumab compared to the 3 mg/mL
composition 1:1 Mix (F26) and to a diluted form of commercial
Avastint (F18; FIG. 67).
Stability of the F19 Composition
[0525] Throughout the 24-week study, composition F19 displayed the
best stability among all of the compositions assayed. Throughout
the study, all F19 compositions remained visually clear and
maintained targeted pH values. This section highlights the
stability profile of this composition.
[0526] By AEX-HPLC analysis, the F19 composition maintained
comparable Antagonist A purity throughout 24 weeks at both
4.degree. C. and 25.degree. C. (FIG. 68). However, when stored at
37.degree. C., the purity of Antagonist A was approximately 5%
lower by the second week (FIG. 68). This trend at 37.degree. C.
continued over the next 12 weeks, as Antagonist A purity dropped by
approximately 20% compared to the other storage conditions for
Antagonist A (FIG. 68).
[0527] WCX-HPLC analysis revealed a correlation between the storage
temperature and the rate of bevacizumab degradation in the F19
composition. After 2 weeks at 37.degree. C., bevacizumab purity
dropped approximately 10% compared to 4.degree. C. samples (FIG.
69). This trend continued up to 12 weeks, where the purity of the
bevacizumab stored at 37.degree. C. dropped approximately 50%
compared to 4.degree. C. (FIG. 69). Storing at 25.degree. C.
maintained comparable purity to 4.degree. C. up to 4 weeks (FIG.
42). However, by the eighth week, the 25.degree. C. samples
suffered a 7% drop in purity relative to the 4.degree. C. samples
(FIG. 69). The increased degradation of bevacizumab stored at
25.degree. C. continued for the rest of the 24 weeks of the study,
where at the end of which bevacizumab purity was approximately 20%
lower than samples stored at 4.degree. C. (FIG. 69). Storage at
4.degree. C. appeared to maintain comparable purity to starting
values throughout the 24 weeks of the study (FIG. 69).
[0528] Composition F19 prevented additional soluble aggregation or
fragmentation of Antagonist A comparable to starting values by
SE-HPLC (FIG. 70). Bevacizumab in F19 stored at 37.degree. C.
maintained comparable purity to storage at 4.degree. C. and
25.degree. C. for up to 2 weeks, after which soluble aggregation
developed by 4 weeks (FIG. 71). Bevacizumab purity was maintained
for up to 8 weeks at 25.degree. C. before significant soluble
aggregation developed by 12 weeks (FIG. 71). At 4.degree. C.,
bevacizumab maintained purity values comparable to the initial time
point for 24 weeks (FIG. 71).
[0529] Contrasts of purity between Antagonist A and bevacizumab
were seen when comparing composition F19 to compositions comprising
only Antagonist A or bevacizuimab. From 2 to 8 weeks at 37.degree.
C., Composition F25 maintained 5-8% higher Antagonist A purity than
F19 by AEX-HPLC analysis. However, by Week 12, both compositions
dropped to similar purity levels (FIG. 72). Furthermore, at
4.degree. C. and 25.degree. C., both compositions maintained
comparable purity levels (FIG. 72). By SE-HPLC, the composition F12
appeared better than F19 at each storage condition with the
greatest difference seen at 4.degree. C., although some assay
variability was observed (FIG. 73).
[0530] Formulating bevacizumab in F19 provided better stability
compared to a diluted form of commercial Avastin.RTM. (F18). Based
on WCX-HPLC, F19 stabilized bevacizumab better than F18 at
25.degree. C. and especially at 37.degree. C., revealing an 8%-11%
improvement from 2-12 weeks (FIG. 74). Similarly, SE-HPLC analysis
showed better prevention of aggregation or fragmentation of
bevacizumab compared to F18 stored at 37.degree. C. (FIG. 75).
[0531] Based on the data collected over the 24 weeks of stability
testing, it was determined that F19 is the most stable composition
of Antagonist A and bevacizumab. Among the compositions tested, F19
helped stabilize both the 3 mg/mL Antagonist A and 12.5 mg/mL
bevacizumab when stored at 4.degree. C. for up to at least 24
weeks. Also, the purity of both Antagonist A and bevacizumab in the
F19 composition was maintained for up to at least 4 weeks at
25.degree. C.
Example 3
Biological Activity of Composition Comprising Ranibizumab and
Antagonist a
[0532] The purpose of this study was to evaluate the biological
activity of a composition comprising both ranibizumab and
Antagonist A, as compared to Lucentis.RTM. and Antagonist A alone.
The activity was measured via the level of gene expression, using
real-time PCR, as a function of inhibition of VEGF and PDGF-BB
binding to their respective cellular receptors. Three different
ranibizumab/Antagonist A compositions were analyzed: F6, F8, and
F11 (see Example 1). The compositions had been stored at 4.degree.
C. for 12 months prior to their use in this study.
[0533] Ranibizumab anti-VEGF activity, alone or present in a
composition also comprising Antagonist A, was determined by its
ability to inhibit VEGF induction of the Tissue Factor (TF) gene in
human umbilical vein endothelial cells (HUVEC). The samples were
analyzed in triplicate and all data normalized to that obtained for
the VEGF only treatment. As shown in FIG. 76, the anti-VEGF EC50
(nM) values determined for the all compostions and for
Lucentis.RTM. alone were identical within a 95% confidence
interval.
[0534] Antagonist A anti-PDGF activity, alone or present in a
composition also comprising ranibizumab, was determined by its
ability to inhibit PDGF-BB induction of BTG2 gene expression in 3T3
fibroblast cell. The samples were analyzed in duplicate and all
data normalized to that obtained for the PDGF-BB only treatment. As
shown in FIG. 77, the anti-PDGF EC50 (nM) values determined for all
compositions and for Antagonist A alone were identical within a 95%
confidence interval. These results demonstrate that a composition
comprising both ranibizumab and Antagonist A shows activity for
each agent for at least 12 months when stored at 4.degree. C.
Example 4
Effect of Storage Conditions on the Stability of Compositions
Comprising Antagonist A and Ranibizumab
[0535] The stability of Antagonist A and ranibizumab in various
compositions was examined using subvisible particle analysis to
evaluate the effects of different storage temperature and different
storage containers. Subvisible particle analysis was performed for
Antagonist A (30 mg/mL), ranibizumab (10 mg/mL and 40 mg/mL), and
various combinations of Antagonist A and ranibizumab by micro-flow
imaging (MFI). A total of five separate compositions were analyzed
following different storage conditions to evaluate the effects of
storage temperature (5.degree. C. and 30.degree. C. for 4 hours)
and storage container (2 cc vials and 1 mL syringes) on the
subvisible particle count for each formulation. The MFI results for
each sample were presented in particular particle size ranges
(including total, .gtoreq.2 .mu.m, .gtoreq.5 .mu.m, .gtoreq.10
.mu.m and .gtoreq.25 .mu.m). Some relative correlation of particle
counts was observed for different samples stored under the same
conditions.
Materials
[0536] The following Antagonist A and ranibizumab compositions were
used in the study: [0537] (1) 30 vials containing 0.23 mL of 30
mg/mL Antagonist A in 10 mM sodium phosphate and 150 mM sodium
chloride, pH 7.3 (Composition F27). [0538] (2) 9 vials containing
0.5 mL of 10 mg/mL ranibizumab in 10 mM histidine HCl, 10%
.alpha.,.alpha.-trehalose and 0.01% polysorbate 20, pH 5.5
(Composition F28; Genentech, South San Francisco, Calif.). [0539]
(3) 7 vials containing 0.5 mL of 40 mg/mL ranibizumab in 10 mM
histidine HCl, 10% .alpha.,.alpha.-trehalose and 0.01% polysorbate
20, pH 5.5 (Composition F29; Genentech, South San Francisco,
Calif.).
[0540] The container materials used for composition preparation are
listed in Table 7.
TABLE-US-00011 TABLE 7 Container materials used in the sample
preparations Item Description Vendor Cat # 5 cc vials.sup..dagger.
Type 1 borosilicate glass, 20 mm finish Schott 68000344 2 cc
vials.sup..dagger. Type 1 borosilicate glass, 13 mm finish Schott
68000314 13 mm vial stoppers FluroTec coated 13 mm serum stopper
West 19700004 13 mm Aluminum Aluminum crimp seal with Flip-Off West
54130229 seal cap 1 mL syringe Luer-Lok Tip Sterile Syringe BD
309628 Syringe stopper Bromobutyl formulation, 4023/50 West Gray
1000 .mu.L Barrier tip* pre-sterile, natural polypropylene Neptune
BT1000 1000 .mu.L tips 25 G 11/2 needles precisionGlide needle BD
305127 .sup..dagger.Vials rinsed with Milli-Q water and dried
before use *Recommended for use with MFI instrument by Protein
Simple
Composition Preparation
[0541] In order to prepare the compositions examined in this study,
vials of the same sample, i.e., Antagonist A or ranibizumab, were
pooled together. In this process, 30 vials of 30 mg/mL Antagonist A
(0.20 mL/vial) were pooled into a 5 cc glass vial, 7 vials of 10
mg/mL ranibizumab (0.5 mL/vial) were pooled into a separate 5 cc
glass vial, and 7 vials of 40 mg/mL ranibizumab (0.5 mL/vial) were
pooled into a third 5 cc glass vial. Although the vials of 30 mg/mL
Antagonist A were intended to contain 0.23 mL, only .about.0.2 mL
was recovered per each vial when pooling. Pooling was performed by
removing the cap from each vial and transferring the contents via
pipette in an aseptic manner. Two additional samples were prepared
in clean glass vials with various combinations of the pooled
materials. Table 8 details the contents for each of the five
samples prepared for this study. To ensure sample cleanliness and
to prevent particle contamination, all pooling and sample
preparations were performed in a class 100 Biological Safety
Cabinet (Nuaire NU-425-600).
TABLE-US-00012 TABLE 8 Composition matrix for MFI analysis Sample
Containers Fill volume per Composition Description filled container
F27 30 mg/mL Ant. A 3 vials and 0.5 mL 2 syringes F28 10 mg/mL 3
vials and 0.5 mL Ranibizumab 2 syringes F29 40 mg/mL 3 vials and
0.5 mL Ranibizumab 2 syringes F30 50% F27 and 3 vials and 0.5 mL
50% F28 2 syringes (by volume) F31 80% F27 and 2 vials and 0.5 mL
20% F29 2 syringes* (by volume) *Not enough volume of F31 was
available to fill one vial; thus only two vials were filled for
this formulation. "Ant. A" is Antagonist A
[0542] In this process, each sample was prepared in a total of two
1 mL syringes and three 2 cc glass vials at a 0.5 mL fill volume,
except for F31, which was prepared in two syringes and two vials.
The various compositions were prepared individually to allow for
precise time point analyses on the MFI instrument. After
preparation, each container was fitted with a stopper, and the
samples were subjected to stability-study conditions.
Storage Conditions
[0543] Samples of each composition were stored at either 5.degree.
C. or 30.degree. C. for 4 hours, in either vials or syringes, to
determine the affects of storage temperature and container type on
the levels of subvisible particles. T=0 analysis was performed on
samples in glass vials immediately after filling. The temperature
conditions and analysis time points for this study are shown in
Table 9.
TABLE-US-00013 TABLE 9 Temperature conditions and analysis time
points Sample Type Storage Temperature Time Point(s) Compositions
in vials 5.degree. C. and 30.degree. C. 0 and 4 hours Compositions
in syringes 5.degree. C. and 30.degree. C. 4 hours
Analytical Analysis and Data Processing
[0544] Size measurements and subvisible particle counts were
collected using an MFI instrument from Brightwell Technologies,
model #DPA-4200. 0.5 mL of each sample was directly applied using a
pipette tip via an inlet port mounted at top of the flow cell for
analysis. In this process, the flow cell was purged with 0.17 mL of
sample, thus affording approximately 0.30 mL for particle
evaluation.
[0545] Subtractions were applied to the MFI data to reduce the
number of air bubbles and non-proteinaceous particles included in
the total particle count. In this process, stuck particles, slow
moving particles, and bubble-like particles with a high circularity
were removed from the data in an attempt to isolate and evaluate
the oligonucleotide or proteinaceous particles in each sample. Edge
particles were also removed in this subtraction, so that the
properties of each particle could be properly screened.
[0546] Results from MFI analysis were obtained as particle counts
per sample. These data were converted to units of particles per mL
of sample by dividing the acquired particle count by the exact
volume analyzed (approximately 0.30 mL). The values of particles
per mL of sample were rounded to the nearest integer.
Results and Discussion
[0547] Table 10 summarizes the results of MFI analysis for the five
compositions. F27 to F31, analyzed in this study. Both the raw and
subtracted MFI data for the compositions under each storage
condition are presented in terms of total particle count/mL, as
well particle counts/mL at particle sizes of .gtoreq.2 .mu.m,
.gtoreq.5 .mu.m, .gtoreq.8 .mu.m, .gtoreq.10 .mu.m and .gtoreq.25
.mu.m. Various results were observed during different temperature
and container conditions.
TABLE-US-00014 TABLE 10 MFI results of Compositions F27 to F31
stored at varying conditions Raw MFI data (particles/mL) Subtracted
MFI data (particles/mL) Comp. Condition Total .gtoreq.2 .mu.m
.gtoreq.5 .mu.m .gtoreq.10 .mu.m .gtoreq.25 .mu.m Total .gtoreq.2
.mu.m .gtoreq.5 .mu.m .gtoreq.10 .mu.m .gtoreq.25 .mu.m water None
498 239 39 13 0 318 141 23 7 0 F27 T = 0 30482 5046 531 75 20 27185
4552 406 69 16 5.degree. C. in vial 29050 4466 518 49 3 25380 3749
200 23 0 (4 hrs) 30.degree. C. in vial 29748 4322 321 46 3 26192
3870 262 46 3 (4 hrs) 5.degree. C. in syringe 59955 15995 2835 226
3 51408 12865 1380 72 3 (4 hrs) 30.degree. C. in syringe 60351
16181 2638 265 3 51041 12305 983 36 0 (4 hrs) F28 T = 0 22788 4231
193 23 3 20399 3788 167 20 3 5.degree. C. in vial 29902 5990 623
151 26 26989 5155 406 102 20 (4 hrs) 30.degree. C. in vial 26553
5322 531 102 13 24207 4811 416 82 10 (4 hrs) 5.degree. C. in
syringe 20648 3490 210 43 13 18649 3188 197 43 13 (4 hrs)
30.degree. C. in syringe 54082 11554 1583 292 52 48908 10555 1265
249 49 (4 hrs) F29 T = 0 17889 3598 426 69 7 16398 3342 383 49 3
5.degree. C. in vial 37190 7956 1268 216 36 33965 7396 1222 213 33
(4 hrs) 30.degree. C. in vial 68393 14068 2212 210 23 62373 13442
2127 206 23 (4 hrs) 5.degree. C. in syringe 57917 12489 2066 380 39
51027 10416 1774 338 36 (4 hrs) 30.degree. C. in syringe 42190 9405
1462 220 13 38753 8923 1396 216 13 (4 hrs) F30 T = 0 58241 14759
2320 383 36 49908 11708 1065 236 36 5.degree. C. in vial 80979
19770 2681 315 49 79210 18000 1616 197 33 (4 hrs) 30.degree. C. in
vial 76893 17905 1891 151 13 67033 15123 1167 111 13 (4 hrs)
5.degree. C. in syringe 90128 22820 2972 279 29 76392 17708 1104 72
13 (4 hrs) 30.degree. C. in syringe 84961 20723 2123 141 26 73583
16994 1232 111 20 (4 hrs) F31 T = 0 69978 19046 3161 310 27 53929
9712 722 78 17 5.degree. C. in vial N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A (4 hrs)* 30.degree. C. in vial 401875 132486 25652 3883 239
324874 107470 16198 3277 233 (4 hrs).sup..DELTA. 5.degree. C. in
syringe 103463 28893 4832 683 89 87777 22848 2578 290 41 (4 hrs)
30.degree. C. in syringe 104495 26232 3628 288 23 90518 22565 2048
111 23 (4 hrs) *Not enough volume of F31 was available to analyze
this particular condition. .sup..DELTA.Due the low volume for F31,
this sample was prepared from a portion of F31 that was initially
drawn into a syringe.
The temperature at T=0 was room temperature.
[0548] The subtracted MFI results for Compositions F27 to F31,
after the different storage conditions, are graphically displayed
as histograms in FIGS. 79 to 83, respectively. These histograms
present the particle counts for each sample at various size ranges,
including 1 to 2 .mu.m, 2 to 5 .mu.m, 5 to 10 .mu.m, 10 to 25
.mu.m, 25 to 50 .mu.m, 50 to 75 .mu.m and 75 to 100 .mu.m. These
Figs. also show varying results for the compositions following
different storage conditions.
[0549] FIG. 84 compares the subtracted MFI results for each sample
at the different storage conditions. In this Fig., particle counts
were evaluated at 1 to 2 .mu.m, 2 to 5 .mu.m, 5 to 10 .mu.m, 10 to
25 .mu.m, 25 to 50 .mu.m and 50 to 75 .mu.m. The high particle
counts observed for F31 in a glass vial following 4 hours at
30.degree. C. may result from sample handling issues. However,
additional sample was not available for re-analysis.
Conclusion
[0550] The execution of particle analysis for Antagonist A,
ranibizumab and various combinations of Antagonist A and
ranibizumab was performed by MFI. A total of 24 different samples
of 5 compositions were analyzed in this study following 4 hours of
storage at 5.degree. C. and 30.degree. C. in either 2 cc glass
vials or 1 mL syringes. The results for each sample were presented
in particular particle size ranges including .gtoreq.2 .mu.m,
.gtoreq.5 .mu.m, .gtoreq.10 .mu.m, .gtoreq.25 .mu.m and total
particle counts. No considerable differences were observed;
however, higher particle counts were detected for F31 in a glass
vial following 30.degree. C. storage.
Example 5
Synthesis of Antagonist A
[0551] An iterative chemical synthesis of the 32-mer
oligonucleotide of Antagonist A was performed on a solid phase
inverted deoxyribothymidine controlled pore glass (CPG) support
using a flow through reactor design. The oligonucleotide synthesis
process was comprised of four chemical reactions carried out in the
following sequence: (a) deblocking of the dimethyoxytrityl (DMT)
protected nucleoside or nascent oligonucleotide (detritylation);
(b) activation and coupling of the incoming phosphoramidite
(amidite); (c) oxidation of the resultant phosphite triester to the
pentavalent phosphate linkage; and (d) capping of oligonucleotide
chains that failed to successfully couple.
[0552] Starting with an inverted thymidine CPG support
(3'-DMT-5'-dT-CPG), the four steps above were repeated to add
phosphoramidites in the order of the sequence until the desired
oligonucleotide, terminating in the hexylamino linker, was
synthesized. The internal hexaethylene glycol spacers were coupled
in the same manner as the other phosphoramidites.
[0553] The first step in the cycle involved removal of the
dimethyoxytrityl protecting group on the terminal hydroxyl group of
the nascent oligonucleotide chain. This was achieved by treating
the DMT protected oligonucleotide on CPG with a solution of
dichloroacetic acid in dichloromethane. This reaction produced the
unprotected terminal hydroxyl group. The cleaved DMT group was
removed with the dichloroacetic acid/dichloromethane (DCA/DCM)
solvent. The CPG was then washed with acetonitrile (ACN).
[0554] The second step involved activation of the incoming
phosphoramidite with ethylthiotetrazole (ETT) to produce a species
that would quickly couple with the terminal hydroxyl group produced
in the previous step. The resultant phosphite triester was washed
with ACN to remove activator and unreacted phosphoramidite
[0555] The third step was oxidation of the newly formed phosphite
triester to the pentavalent phosphate. This was accomplished by
reacting the phosphite triester with a mixture of iodine and
pyridine in water. Unused oxidant was washed from the CPG with
ACN.
[0556] The fourth step involved capping of any unreacted hydroxyls
that had failed to couple. The CPG was treated with a mixture of
CAP NMI (N-methylimidazole in ACN) and CAP ALA (acetic anhydride,
2,6-lutidine, ACN). These reagents were washed from the CPG with
ACN.
[0557] This cycle of four reactions was repeated until an
oligonucleotide of the correct length and sequence was assembled on
the solid support. The last phosphoramidite (hexylamino linker at
the 5' terminus of the oligonucleotide) was reacted in the same
fashion as the other phosphoramidites used in the synthesis;
however, this linker was not capped.
[0558] The oligonucleotide was deprotected and cleaved by treating
the solid support, containing the crude synthesized
oligonucleotide, with a t-butyl amine/ammonium hydroxide solution.
The CPG was separated from the deprotected and cleaved
oligonucleotide. The purity of the crude fully deprotected
oligonucleotide was determined by analytical anion exchange
chromatography and met a specification of greater than 50%.
[0559] The resultant oligonucleotide was diafiltered against sodium
chloride to remove amine salts.
[0560] A covalent bond was then formed between the primary amine at
the 5' end of the oligonucleotide and the pegylation reagent
(mPEG.sub.2-NHS ester). The reaction was conducted at pH 9 in
sodium borate buffer. The reaction has been demonstrated to be site
specific to the hexylamino linker at the 5' end of the
oligonucleotide using the pegylation conditions described.
[0561] The pegylated oligonucleotide was purified from unconjugated
PEG reagent, unpegylated aptamer, and other by-products by
preparative anion exchange chromatography (AX HPLC). The individual
fractions were analyzed by analytical AX HPLC. Selected fractions
of full length pegylated oligonucleotide were pooled and the
resultant pool was desalted, concentrated, and filtered.
[0562] The resultant Antagonist A was vacuum freeze dried to reduce
the water content.
[0563] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification or listed in any Application Data Sheet are
incorporated herein by reference in their entirety.
[0564] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Sequence CWU 1
1
8519DNAartificialsynthetic aptamer 1caggcuacg
9212DNAartificialsynthetic aptamer 2cgtagagcau ca
1238DNAartificialsynthetic aptamer 3tgatccug
8456DNAartificialsynthetic aptamer 4caggctacga ugcaguuuga
gaagucgcgc aucgtagagc atcagaaatg atcctg 56554DNAartificialsynthetic
aptamer 5caggctacgu gcaguuugag aagucgcgca cgtagagcat cagaaatgat
cctg 54639DNAartificialsynthetic aptamer 6cacaggctac ggcacgtaga
gcatcaccat gatcctgtg 39722DNAartificialsynthetic aptamer
7tgactgtgaa cgttcgagat ga 22814DNAartificialsynthetic aptamer
8tgaacgttcg agat 14912DNAartificialsynthetic aptamer 9aacgttcgag at
121010DNAartificialsynthetic aptamer 10aacgttcgag
101113DNAartificialsynthetic aptamer 11gtgaacgttc gag
131224DNAartificialsynthetic aptamer 12tcgtcgtttt gtcgttttgt cgtt
241318DNAartificialsynthetic aptamer 13gtcgttttgt cgttttgt
181414DNAartificialsynthetic aptamer 14gtcgttttgt cgtt
141546DNAartificialsynthetic aptamer 15aacgttcgag caggctacgg
cacgtagagc atcaccatga tcctgc 461649DNAartificialsynthetic aptamer
16gtgaacgttc gagcaggcta cggcacgtag agcatcacca tgatcctgc
491764DNAartificialsynthetic aptamer 17tgactgtgaa cgttcgagat
gacaggctac ggcacgtaga gcatcaccat gatcctgttt 60tttt
641834DNAartificialsynthetic aptamer 18caggctacgt tcgtagagca
tcaccatgat cctg 341935DNAartificialsynthetic aptamer 19caggctacgt
ttcgtagagc atcaccatga tcctg 352035DNAartificialsynthetic aptamer
20caggcaacgt ttcgttgagc atcaccatga tcctg
352134DNAartificialsynthetic aptamer 21caggcaacgt tcgttgagca
tcaccatgat cctg 342236DNAartificialsynthetic aptamer 22caggcaacgt
tttcgttgag catcaccatg atcctg 362335DNAartificialsynthetic aptamer
23caggctacgt ttcgtagagc atcaccatga tcctg
352435DNAartificialsynthetic aptamer 24caggctacgt ttcgtagagc
atcaccatga tcctg 352536DNAartificialsynthetic aptamer 25caggcgtcgt
tttcgacgag catcaccatg atcctg 362636DNAartificialsynthetic aptamer
26caggcgtcgt cgtcgacgag catcaccatg atcctg
362736DNAartificialsynthetic aptamer 27caggcttcgt cgtcgaagag
catcaccatg atcctg 362836DNAartificialsynthetic aptamer 28caggctacgt
cgtcgtagag catcaccatg atcctg 362935DNAartificialsynthetic aptamer
29caggcaagct ttgcttgagc atcaccatga tcctg
353036DNAartificialsynthetic aptamer 30caggcaagct tttgcttgag
catcaccatg atcctg 36319DNAartificialsynthetic aptamer 31caggctacg
93212DNAartificialsynthetic aptamer 32cgtagagcat ca
12338DNAartificialsynthetic aptamer 33tgatcctg
8349DNAartificialsynthetic aptamer 34caggcuacg
93512DNAartificialsynthetic aptamer 35cgtagagcau ca
12368DNAartificialsynthetic aptamer 36tgatccug
8379DNAartificialsynthetic aptamer 37caggctacg
93812DNAartificialsynthetic aptamer 38cgtagagcat ca
12398DNAartificialsynthetic aptamer 39tgatcctg
8409DNAartificialsynthetic aptamer 40caggcuacg
94112DNAartificialsynthetic aptamer 41cguagagcau ca
12428RNAartificialsynthetic aptamer 42ugauccug
84310DNAartificialsynthetic aptamer 43acaggctacg
10449DNAartificialsynthetic aptamer 44tgatcctgt
94511DNAartificialsynthetic aptamer 45cacaggctac g
114610DNAartificialsynthetic aptamer 46tgatcctgtg
10479DNAartificialsynthetic aptamer 47caggctacg
9489DNAartificialsynthetic aptamer 48tgatccugu
94910DNAartificialsynthetic aptamer 49tgatccugug
10509DNAartificialsynthetic aptamer 50caggctacg
9519DNAartificialsynthetic aptamer 51caggcuacg
95212DNAartificialsynthetic aptamer 52cgtagagcat ca
125312DNAartificialsynthetic aptamer 53cgtagagcau ca
12549DNAartificialsynthetic aptamer 54caggcuacg
95512DNAartificialsynthetic aptamer 55cguagagcau ca
12568DNAartificialsynthetic aptamer 56ugauccug
8578DNAartificialsynthetic aptamer 57tgatccug
85811DNAartificialsynthetic aptamer 58cacaggctac g
115910DNAartificialsynthetic aptamer 59tgatcctgtg
106010DNAartificialsynthetic aptamer 60tgatccugug
106111DNAartificialsynthetic aptamer 61cacaggcuac g
11628DNAartificialsynthetic aptamer 62tgatccug
8638DNAartificialsynthetic aptamer 63tgatcctg
86411DNAartificialsynthetic aptamer 64cccaggctac g
116510DNAartificialsynthetic aptamer 65tgatcctggg
106610DNAartificialsynthetic aptamer 66tgatcctggg
106710DNAartificialsynthetic aptamer 67tgatcctggg
106810DNAartificialsynthetic aptamer 68tgatcctggg
106910DNAartificialsynthetic 69aacgttcgag
107087RNAartificialsynthetic aptamer 70gggaaaagcg aaucauacac
aagaucgcca ggagcaaagu cacggaggag uggggguacg 60aaugcuccgc cagagaccaa
ccgagaa 877188RNAartificialsynthetic aptamer 71gggaaaagcg
aaucauacac aagaccggga acucggauuc uucgcaugug gaugcgauca 60guaugcuccg
ccagagacca accgagaa 887288RNAartificialsynthetic aptamer
72gggaaaagcg aaucauacac aagaccggga acucggauuc uucacaugug gaugugauca
60guaugcuccg ccagagacca accgagaa 887388RNAartificialsynthetic
aptamer 73gggaaaagcg aaucauacac aagaccggaa acucggauuc uucgcaugug
gaugcgauca 60guaugcuccg ccagagacca accgagaa
887488RNAartificialsynthetic aptamer 74gggaaaagcg aaucauacac
aagagagugg aggagguaug uaugguuugu gcgucuggug 60cggugcuccg ccagagacca
accgagaa 887529DNAArtificial Sequenceanti-PDGF aptamer 75caggctacgc
gtagagcatc atgatcctg 2976124PRTArtificial SequenceHumanized
anti-VEGF antibody heavy chain variable domain 76Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Xaa Phe Thr Xaa Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Xaa Tyr Tyr Gly Xaa
Ser His Trp Tyr Phe Asp Asn 100 105 110 Val Val Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 77118PRTArtificial SequenceHumanized
anti-VEGF antibody heavy chain variable domain 77Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser
Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu 115
7811PRTMus musculus 78Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1
5 10 797PRTMus musculus 79Phe Thr Ser Ser Leu His Ser 1 5 809PRTMus
musculus 80Gln Gln Tyr Ser Thr Val Pro Trp Thr 1 5
81108PRTArtificial SequenceHumanized anti-VEGF antibody light chain
variable domain 81Asp Ile Gln Xaa Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser
Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu
His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
82110PRTArtificial SequenceHumanized anti-VEGF antibody light chain
variable domain 82Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser
Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu
His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110
8310PRTArtificial SequenceVariant hypervariable region of CDRH1
83Gly Tyr Asp Phe Thr His Tyr Gly Met Asn 1 5 10 8414PRTArtificial
SequenceVariant hypervariable region of CDRH3 84Tyr Pro Tyr Tyr Tyr
Gly Thr Ser His Trp Tyr Phe Asp Val 1 5 10 85458PRTArtificial
SequenceVEGF-Trap - dimeric fusion polypeptide comprising two
fusion polypeptides 85Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu
Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser
Gly Ser Asp Thr Gly Arg Pro 20 25 30 Phe Val Glu Met Tyr Ser Glu
Ile Pro Glu Ile Ile His Met Thr Glu 35 40 45 Gly Arg Glu Leu Val
Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr 50 55 60 Val Thr Leu
Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys 65 70 75 80 Arg
Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr 85 90
95 Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
100 105 110 Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr
Ile Ile 115 120 125 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu
Ser Val Gly Glu 130 135 140 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
Glu Leu Asn Val Gly Ile 145 150 155 160 Asp Phe Asn Trp Glu Tyr Pro
Ser Ser Lys His Gln His Lys Lys Leu 165 170 175 Val Asn Arg Asp Leu
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 180 185 190 Leu Ser Thr
Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 195 200 205 Tyr
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 210 215
220 Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys
225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 340
345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu 355 360 365 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 450 455
* * * * *